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Hand, Foot and Mouth Disease (HFMD) is usually a self-limiting, mild childhood disease that is caused mainly by Coxsackie virus A16 (CVA16) and Enterovirus A71 (EV-A71), both members of the Picornaviridae family. However, recurring HFMD outbreaks and epidemics due to EV-A71 infection in the Western Pacific region, and the propensity of EV-A71 strains to cause severe neurological complications have made this neurotropic virus a serious public health concern in afflicted countries. High mutation rate leading to viral quasispecies combined with frequent intra- and inter-typic recombination events amongst co-circulating EV-A71 strains have contributed to the great diversity and fast evolution of EV-A71 genomes, making impossible any accurate prediction of the next epidemic strain. Comparative genome sequence analyses and mutagenesis approaches have led to the identification of a number of viral determinants involved in EV-A71 fitness and virulence. These viral determinants include amino acid residues located in the structural proteins of the virus, affecting attachment to the host cell surface, receptor binding, and uncoating events. Critical residues in non-structural proteins have also been identified, including 2C, 3A, 3C proteases and the RNA-dependent RNA polymerase. Finally, mutations altering key secondary structures in the 5' untranslated region were also found to influence EV-A71 fitness and virulence. While our current understanding of EV-A71 pathogenesis remains fragmented, these studies may help in the rational design of effective treatments and broadly protective vaccine candidates.

Hand, foot and mouth disease (HFMD) is a common infectious disease in western Asia area and the full range of the long-term sequelae of HFMD remains poorly described. We conducted a retrospective hospital-based cohort study of HFMD patients with central nervous system (CNS) complications caused by EV-A71 or CV-A16 between 2010 and 2016. Patients were classified into three groups, including CNS only, autonomic nervous system (ANS) dysregulation, and cardiorespiratory failure. Neurologic examination, neurodevelopmental assessments, Magnetic Resonance Imaging (MRI) and lung function, were performed at follow up. Of the 176 patients followed up, 24 suffered CNS only, 133 ANS dysregulation, and 19 cardiorespiratory failure. Median follow-up period was 4.3 years (range [1.4-8.3]). The rate of neurological abnormalities was 25% (43 of 171) at discharge and 10% (17 of 171) at follow-up. The rates of poor outcome were significantly different between the three groups of complications in motor (28%, 38%, 71%) domain (p=0.020), but not for cognitive (20%, 24%, 35%), language (25%, 36%, 41%) and adaptive (24%, 16%, 26%) domains ( p  = 0.537, p  = 0.551, p  = 0.403). For children with ventilated during hospitalization, 41% patients (14 of 34) had an obstructive ventilatory defect, and one patient with scoliosis had mixed ventilatory dysfunction. Persistent abnormalities on brain MRI were 0% (0 of 7), 9% (2 of 23) and 57% (4 of 7) in CNS, ANS and cardiorespiratory failure group separately. Patients with HFMD may have abnormalities in neurological, motor, language, cognition, adaptive behaviour and respiratory function. Long-term follow-up programmes for children's neurodevelopmental and respiratory function may be warranted.

Enterovirus 71 can cause severe hand, foot, and mouth disease (HFMD) in children. However, little is known about the mechanism of inflammatory disorders caused by EV71 infection and why severe cases are mainly children aged under-three. In current study, using mRNA microarray assay, the differential expression of Placenta-specific 8 (PLAC8) was identified in mice brain. In addition, we found that PLAC8 expression was down-regulated with age in mice lung tissues and human peripheral blood. Then, we further proved that PLAC8 could promote inflammation progress and disturb Th1/Th2/Th17/Treg related cytokines release after EV71 infection using PLAC8 plasmid over-expressed neonatal mouse model. Our data suggest that PLAC8 might play a crucial role in Th cell differentiation and inflammatory damage caused by EV71 infection in infants. Thus, our findings would help understand the causes of severe inflammatory injury in infants during EV71 infection, and provide new insights into the prevention and control of severe HFMD., (Copyright © 2021. Published by Elsevier Inc.)

ABSTRACT Enterovirus A71 (EV-A71) can cause hand, foot and mouth disease with neurological and systemic complications, most frequently affecting children and infants. We describe a cis- acting replication element ( cre ) with a conserved stem-loop structure within the EV-A71 2C-coding region. By site-directed mutagenesis and reverse genetics using the EV-A71 full-length genome and the EV-A71 replicon containing the firefly luciferase reporter gene in place of the P1 region, the stem-loop structure and the AAACA in the loop of the cre were confirmed to be required for the EV-A71 replication phenotype. EV-A71 genomes containing a mutation at the first or third A residue of AAACA could not be recovered. Insertion of a wild-type cre from EV-A71 or poliovirus in the 5'UTR led to successful recovery of the replication of nonviable mutants. Furthermore, the cre mutants showed lower binding capacity with the host cellular factor IGF2BP2, knockdown of which resulted in a significant decrease in EV-A71 production. All the available evidence shows the location independence but functional importance of the interaction of the cre with the cellular host for efficient production of EV-A71, contributing to the growing body of knowledge regarding picornavirus cre s.

Positive-strand RNA virus evolution is partly attributed to the process of recombination. Although common between closely genetically related viruses, such as within species of the Enterovirus genus of the Picornaviridae family, inter-species recombination is rarely observed in nature. Recent studies have shown recombination is a ubiquitous process, resulting in a wide range of recombinant genomes and progeny viruses. While not all recombinant genomes yield infectious progeny virus, their existence and continued evolution during replication have critical implications for the evolution of the virus population. In this study, we utilised an in vitro recombination assay to demonstrate inter-species recombination events between viruses from four enterovirus species, A-D. We show that inter-species recombinant genomes are generated in vitro with polymerase template-switching events occurring within the virus polyprotein coding region. However, these genomes did not yield infectious progeny virus. Analysis and attempted recovery of a constructed recombinant cDNA revealed a restriction in positive-strand but not negative-strand RNA synthesis, indicating a significant block in replication. This study demonstrates the propensity for inter-species recombination at the genome level but suggests that significant sequence plasticity would be required in order to overcome blocks in the virus life cycle and allow for the production of infectious viruses.

Enterovirus D68 (EV-D68) is associated with a broad spectrum of illnesses, including mild to severe acute respiratory illness (ARI) and acute flaccid myelitis (AFM). Enteroviruses, including EV-D68, are typically detected in the United States during late summer through fall, with year-to-year fluctuations. Before 2014, EV-D68 was infrequently reported to CDC (1). However, numbers of EV-D68 detection have increased in recent years, with a biennial pattern observed during 2014-2018 in the United States, after the expansion of surveillance and wider availability of molecular testing. In 2014, a national outbreak of EV-D68 was detected (2). EV-D68 was also reported in 2016 via local (3) and passive national (4) surveillance. EV-D68 detections were limited in 2017, but substantial circulation was observed in 2018 (5). To assess recent levels of circulation, EV-D68 detections in respiratory specimens collected from patients aged <18 years* with ARI evaluated in emergency departments (EDs) or admitted to one of seven U.S. medical centers † within the New Vaccine Surveillance Network (NVSN) were summarized. This report provides a provisional description of EV-D68 detections during July-November in 2018, 2019 and 2020, and describes the demographic and clinical characteristics of these patients. In 2018, a total of 382 EV-D68 detections in respiratory specimens obtained from patients aged <18 years with ARI were reported by NVSN; the number decreased to six detections in 2019 and 30 in 2020. Among patients aged <18 years with EV-D68 in 2020, 22 (73%) were non-Hispanic Black (Black) persons. EV-D68 detections in 2020 were lower than anticipated based on the biennial circulation pattern observed since 2014. The circulation of EV-D68 in 2020 might have been limited by widespread COVID-19 mitigation measures; how these changes in behavior might influence the timing and levels of circulation in future years is unknown. Ongoing monitoring of EV-D68 detections is warranted for preparedness for EV-D68-associated ARI and AFM., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Janet A. Englund reports institutional research support from AstraZeneca, Merck & Co., Pfizer Inc., and GlaxoSmithKline plc; consulting fees from Sanofi Pasteur, Meissa Vaccines Incorporated, AstraZeneca, and Teva Pharmaceutical Industries Ltd.; and unpaid membership on the publication committees for the Infectious Diseases Society of America and the Pediatric Infectious Diseases Society. Christopher J. Harrison reports institutional grant support from GlaxoSmithKline plc and Pfizer Inc., for vaccine studies and from Merck & Co. for a study of antibiotic resistance, and royalties from UpToDate for editing chapter on rotavirus. Natasha B. Halasa reports institutional support from Sanofi Pasteur and Quidel Corporation. Geoffrey A. Weinberg reports honoraria as a consultant to ReViral Ltd, and as an author of textbook chapters in the Merck Manual. No other potential conflicts of interest were disclosed.

Background: Because of limited infrastructure and skilled human capital, the etiology of meningitis is rarely identified in developing countries like Ethiopia. This results in unnecessary antibiotics use, economic crisis, hospitalization, and related nosocomial infections. Thus, we aimed to assess the epidemiology of human enteroviruses (HEVs) among clinically suspected meningitis cases in Addis Ababa, Ethiopia., Method: A cross-sectional study was conducted from January to August 2020 at selected Hospitals in Addis Ababa, Ethiopia. Reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted on cerebrospinal fluid (CSF) collected from 146 clinically suspected meningitis and bacterial culture-negative patients. SPSS v 21.0 was used for data analysis and bivariate correlation was done for the association between variables of interest., Results: HEVs were detected in 39 (26.7%) of the 146 clinically suspected meningitis cases. Most of the HEVs cases 28 (71.9%) were detected in younger-aged infants less than 1 year. The most commonly observed clinical manifestations were vomiting (75.5%) followed by fever (56.8%) and impaired consciousness or irritability (50.7%). The mean length of hospital stay for patients with enteroviral meningitis was 9 days. Many patients with HEVs were recovered with sequelae (46.2%), and HEVs has contributed for one out of the nine meningeal deaths (11.1%)., Conclusions: HEVs were found to be the commonest cause of morbidity and mortality in all age groups. Many of the patients were mistreated with antibiotics and hospitalized. The detection of HEVs in 26.7% of clinically suspected meningitis cases indicated the need for molecular tests in investigating the etiology of meningitis. Therefore, we suggest the introduction of molecular tests as a routine practice in referral hospitals and the need to further characterize circulating HEVs strains., Competing Interests: The authors have declared that no competing interests exist.

Although epidemics of hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) have occurred worldwide, the Asia-Pacific region has seen large sporadic outbreaks with many severe neurological cases. This suggests that the virulence of the circulating viruses fluctuates in each epidemic and that HFMD outbreaks with many severe cases occur when highly virulent viruses are circulating predominantly, which has not been experimentally verified. Here, we analyzed 32 clinically isolated strains obtained in Japan from 2002 to 2013, along with 27 Vietnamese strains obtained from 2015 to 2016 that we characterized previously using human SCARB2 transgenic mice. Phylogenetic analysis of the P1 region classified them into five clades belonging to subgenogroup B5 (B5-I to B5-V) and five clades belonging to subgenogroup C4 (C4-I to C4-V) according to the epidemic year and region. Interestingly, clades B5-I and B5-II were very virulent, while clades B5-III, B5-IV, and B5-V were less virulent. Clades C4-II, C4-III, C4-IV, and C4-V were virulent, while clade C4-I was not. The result experimentally showed for the first time that several clades with different virulence levels emerged one after another. The experimental virulence evaluation of circulating viruses using SCARB2 transgenic mice is helpful to assess potential risks of circulating viruses. These results also suggest that a minor nucleotide or amino acid substitution in the EV-A71 genome during circulation causes fluctuations in virulence. The data presented here may increase our understanding of the dynamics of viral virulence during epidemics. IMPORTANCE Outbreaks of hand, foot, and mouth disease (HFMD) with severe enterovirus A71 (EV-A71) cases have occurred repeatedly, mainly in Asia. In severe cases, central nervous system complications can lead to death, making it an infectious disease of importance to public health. An unanswered question about this disease is why outbreaks of HFMD with many severe cases sometimes occur. Here, we collected EV-A71 strains that were prevalent in Japan and Vietnam over the past 20 years and evaluated their virulence in a mouse model of EV-A71 infection. This method clearly revealed that viruses belonging to different clades have different virulence, indicating that the method is powerful to assess the potential risks of the circulating viruses. The results also suggested that factors in the virus genome cause an outbreak with many severe cases and that further studies facilitate the prediction of large epidemics of EV-A71 in the future.

Recently, bovine enterovirus (BEV) has caused several respiratory and gastrointestinal diseases outbreaks in cattle. Monitoring the epidemiological and pathogenic characteristics of this virus is crucial to controlling its spread. We isolated a BEV strain with typical cytopathic effects from the faeces of cows with significant diarrhoeal symptoms in China and observed the viral particles within 20-30 nm through transmission electron microscopy. Then, we designated this strain as HB19-1 in this study. The multistep growth curves showed that the virus propagated well in the MDBK cells. Molecular genetic analysis of VP1 indicated that HB19-1 belonged to the BEV-F1 group. Although the challenged ICR mice did not exhibit typical disease symptoms in animal infection assay, we observed significant pathological damage in the lungs, intestines, and muscle tissues. In summary, we isolated a BEV strain HB19-1 causing severe diarrhoea in cattle and proposed reinforcing the epidemiological surveillance of this virus.

Introduction: Patients with hemoglobinopathies have been reported to have higher rates of pulmonary complications. Few studies have investigated the association between thalassemia and asthma in children., Methods: We used the data of one million individuals randomly selected from the Registry for Beneficiaries of the National Health Insurance Research Database. One thalassemic child was matched with four control children without thalassemia according to sex, birth year, birth season, prematurity, and previous enteroviral infection., Results: A total of 800 hundred thalassemic children and 3200 controls were included. Children with thalassemia had higher rates of developing asthma (41.81 vs 25.70 per 1000 person-years, P < 0.001) than the non-thalassemia controls with an adjusted hazard ratio of 1.37 (95% confidence interval [CI] = 1.19-1.58). Boys in the thalassemia cohort had a significantly higher adjusted incidence hazard ratio (IRR) of asthma than those in the non-thalassemia cohort (adjusted IRR = 1.45, 95% CI = 1.02-1.73). The risk of atopic and nonatopic asthma was higher in the thalassemia cohort than in the non-thalassemia cohort (IRR = 1.3, 1.61, respectively)., Conclusions: Children with thalassemia were more likely to develop asthma. More attention should be paid to the early diagnosis of asthma and prevention of asthma attacks., Competing Interests: The authors have declared that no competing interests exist.

Objective: To evaluate the pathogenicity of a broad range of 11 possible gastroenteritis viruses, by means of statistical relationships with cases vs. controls, or Ct-values, in order to establish the most appropriate diagnostic panel for our general practitioner (GP) patients in the Netherlands (2010-2012)., Methods: Archived stool samples from 1340 cases and 1100 controls were retested using internally controlled multiplex real-time PCRs for putative pathogenic gastroenteritis viruses: adenovirus, astrovirus, bocavirus, enterovirus, norovirus GI and GII, human parechovirus, rotavirus, salivirus, sapovirus, and torovirus., Results: The prevalence of any virus in symptomatic cases and asymptomatic controls was 16.6% (223/1340) and 10.2% (112/1100), respectively. Prevalence of astrovirus (adjusted odds ratio (aOR) 10.37; 95% confidence interval (CI) 1.34-80.06) and norovirus GII (aOR 3.10; CI 1.62-5.92) was significantly higher in cases versus controls. Rotavirus was encountered only in cases. We did not find torovirus and there was no statistically significant relationship with cases for salivirus (aOR 1,67; (CI) 0.43-6.54)), adenovirus non-group F (aOR 1.20; CI 0.75-1.91), bocavirus (aOR 0.85; CI 0.05-13.64), enterovirus (aOR 0.83; CI 0.50-1.37), human parechovirus (aOR 1.61; CI 0.54-4.77) and sapovirus (aOR 1.15; CI 0.67-1.98). Though adenovirus group F (aOR 6.37; CI 0.80-50.92) and norovirus GI (aOR 2.22, CI: 0.79-6.23) are known enteropathogenic viruses and were more prevalent in cases than in controls, this did not reach significance in this study. The Ct value did not discriminate between carriage and disease in PCR-positive subjects., Conclusions: In our population, diagnostic gastroenteritis tests should screen for adenovirus group F, astrovirus, noroviruses GI and GII, and rotavirus. Case-control studies as ours are lacking and should also be carried out in populations from other epidemiological backgrounds., Competing Interests: The authors have declared that no competing interests exist.

New circulating Enterovirus (EV) strains often emerge through recombination. Upsurges of recombinant non-polio enteroviruses (NPEVs) associated with neurologic manifestations such as EVA71 or Echovirus 30 (E30) are a growing public health concern in Europe. Only a few complete genomes of EVs circulating in Spain are available in public databases, making it difficult to address the emergence of recombinant EVs, understand their evolutionary relatedness and the possible implication in human disease. We have used metagenomic (untargeted) NGS to generate full-length EV genomes from CSF samples of EV-positive aseptic meningitis cases in Southern Spain between 2015 and 2018. Our analyses reveal the co-circulation of multiple Enterovirus B (EV-B) types (E6, E11, E13 and E30), including a novel E13 recombinant form. We observed a genetic turnover where emergent lineages (C1 for E6 and I [tentatively proposed in this study] for E30) replaced previous lineages circulating in Spain, some concomitant with outbreaks in other parts of Europe. Metagenomic sequencing provides an effective approach for the analysis of EV genomes directly from PCR-positive CSF samples. The detection of a novel, disease-associated, recombinant form emphasizes the importance of genomic surveillance to monitor spread and evolution of EVs., (© 2021. The Author(s).)

Coxsackievirus A16 (CV-A16) has caused worldwide epidemics of hand, foot, and mouth disease (HFMD) in infants and preschool children. Circular RNAs (circRNAs), a class of noncoding RNA molecules, participate in the progression of viral infectious diseases. Although the function of circRNAs has been a heavily researched topic, their role in CV-A16 infection is still unclear. In this study, the viral effects of CV-A16 on the cellular circRNA transcriptome were investigated using next-generation sequencing technology. The results showed that a total of 8726, 8611, and 6826 circRNAs were identified at 0, 12, and 24 h postinfection, respectively. Moreover, it was found that 1769 and 1192 circRNAs were differentially expressed in at 12 and 24 h postinfection, respectively. The common differentially expressed circRNAs were used for functional annotation analysis, and it was found that the parent genes of differentially expressed circRNAs might be associated with the viral infection process, especially the "Immune system process" in GO analysis and the "Inflammation mediated by chemokine and cytokine signaling pathway" in KEGG analysis. Subsequently, circRNA-miRNA-mRNA regulatory networks were constructed, and the hsa&#95;circ&#95;0004447/hsa-miR-942-5p/MMP2, hsa&#95;circ&#95;0078617/hsa-miR-6780b-5p/MMP2 and hsa&#95;circ&#95;0078617/hsa-miR-5196-5p/MMP2 regulatory axes were identified by enrichment analysis as important networks during the progression of CV-A16 infection. Finally, six dysregulated circRNAs were selected for validation and were verified to be consistent with the sequencing results. Considering all of these results, to the best of our knowledge, this study is the first to present a comprehensive overview of circRNAs induced by CV-A16 infection, and this research demonstrated that a network of enriched circRNAs and circRNA-associated competitive endogenous RNAs (ceRNAs) is involved in the regulation of CV-A16 infection, thereby helping to elucidate the mechanisms underlying CV-A16-host interactions., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Aims/hypothesis: The Diabetes Virus Detection (DiViD) study is the first study to laparoscopically collect pancreatic tissue and purified pancreatic islets together with duodenal mucosa, serum, peripheral blood mononuclear cells (PBMCs) and stools from six live adult patients (age 24-35 years) with newly diagnosed type 1 diabetes. The presence of enterovirus (EV) in the pancreatic islets of these patients has previously been reported., Methods: In the present study we used reverse transcription quantitative real-time PCR (RT-qPCR) and sequencing to characterise EV genomes present in different tissues to understand the nature of infection in these individuals., Results: All six patients were found to be EV-positive by RT-qPCR in at least one of the tested sample types. Four patients were EV-positive in purified islet culture medium, three in PBMCs, one in duodenal biopsy and two in stool, while serum was EV-negative in all individuals. Sequencing the 5' untranslated region of these EVs suggested that all but one belonged to enterovirus B species. One patient was EV-positive in all these sample types except for serum. Sequence analysis revealed that the virus strain present in the isolated islets of this patient was different from the strain found in other sample types. None of the islet-resident viruses could be isolated using EV-permissive cell lines., Conclusions/interpretation: EV RNA can be frequently detected in various tissues of patients with type 1 diabetes. At least in some patients, the EV strain in the pancreatic islets may represent a slowly replicating persisting virus., (© 2021. The Author(s).)

Enterovirus, like the majority of RNA viruses, evolves to survive the changeable environments by a variety of strategies. Here, we showed that HY12 virus evolved to alter its characteristics and pathogenicity by employing a non-synonymous mutation. Analyses of 5'UTR, VP1 and VP2 gene sequences revealed the existence of HY12 virus in an array of mutants defined as quasispecies. The determination of diversity and complexity showed that the mutation rate and complexity of HY12 virus quasispecies increased, while the proportion of HY12 VP1 and VP2 consensus (master) sequences decreased with increasing passages. Synonymous mutation and non-synonymous mutation analysis displayed a positive selection for HY12 quasispecies evolution. A comparison of HY12 virus in different passages demonstrated that HY12 virus altered its characteristic, phenotype, and pathogenicity via non-synonymous mutation. These findings revealed the evolution pattern for HY12 virus, and the alteration of HY12 virus characteristics and pathogenicity by mutation.

Infection by rhinovirus (RV) and enterovirus (EV) in children ranges from asymptomatic infection to severe lower respiratory tract infection (LRTI). This cohort study evaluates the clinical impact of RV/EV species, alone or in codetection with other viruses, in young children with severe LRTI. Seventy-one patients aged less than 5 years and admitted to the Paediatric Intensive Care Unit (PICU) of a reference children's hospital with RV or EV (RV/EV) LRTI were prospectively included from 1/2018 to 3/2020. A commercial PCR assay for multiple respiratory pathogens was performed in respiratory specimens. In 22/71, RV/EV + respiratory syncytial virus (RSV) was found, and 18/71 had RV/EV + multiple viral detections. Patients with single RV/EV detection required invasive mechanical ventilation (IMV) as frequently as those with RSV codetection, whereas none of those with multiple viral codetections required IMV. Species were determined in 60 samples, 58 being RV. No EV-A, EV-C, or EV-D68 were detected. RV-B and EV-B were only found in patients with other respiratory virus codetections. There were not any associations between RV/EV species and severity outcomes. To conclude, RV/EV detection alone was observed in young children with severe disease, while multiple viral codetections may result in reduced clinical severity. Differences in pathogenicity between RV and EV species could not be drawn.

Enterovirus A71 (EV-A71) and many members of the Picornaviridae family are neurotropic pathogens of global concern. These viruses are primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. An animal model of oral infection was developed using transgenic mice expressing human SCARB2 (hSCARB2 Tg), murine-adapted EV-A71/MP4 virus, and EV-A71/MP4 virus with an engineered nanoluciferase gene that allows imaging of viral replication and spread in infected mice. Next-generation sequencing of EV-A71 genomes in the tissues and organs of infected mice was also performed. Oral inoculation of EV-A71/MP4 or nanoluciferase-carrying MP4 virus stably induced neurological symptoms and death in infected 21-day-old weaned mice. In vivo bioluminescence imaging of infected mice and tissue immunostaining of viral antigens indicated that orally inoculated virus can spread to the central nervous system (CNS) and other tissues. Next-generating sequencing further identified diverse mutations in viral genomes that can potentially contribute to viral pathogenesis. This study presents an EV-A71 oral infection murine model that efficiently infects weaned mice and allows tracking of viral spread, features that can facilitate research into viral pathogenesis and neuroinvasion via the natural route of infection. IMPORTANCE Enterovirus A71 (EV-A71), a positive-strand RNA virus of the Picornaviridae , poses a persistent global public health problem. EV-A71 is primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. We present an animal model of EV-A71 infection that enables the natural route of oral infection in weaned and nonimmunocompromised 21-day-old hSCARB2 transgenic mice. Our results demonstrate that severe disease and death could be stably induced, and viral invasion of the CNS could be replicated in this model, similar to severe real-world EV-A71 infections. We also developed a nanoluciferase-containing EV-A71 virus that can be used with this animal model to track viral spread after oral infection in real time. Such a model offers several advantages over existing animal models and can facilitate future research into viral spread, tissue tropism, and viral pathogenesis, all pressing issues that remain unaddressed for EV-A71 infections.

RNA interference (RNAi) is the major antiviral mechanism in plants and invertebrates, but the absence of detectable viral (v)siRNAs in mammalian cells upon viral infection has questioned the functional relevance of this pathway in mammalian immunity. We designed a series of peptides specifically targeting enterovirus A71 (EV-A71)-encoded protein 3A, a viral suppressor of RNAi (VSR). These peptides abrogated the VSR function of EV-A71 in infected cells and resulted in the accumulation of vsiRNAs and reduced viral replication. These vsiRNAs were functional, as evidenced by RISC-loading and silencing of target RNAs. The effects of VSR-targeting peptides (VTPs) on infection with EV-A71 as well as another enterovirus, Coxsackievirus-A16, were ablated upon deletion of Dicer1 or AGO2, core components of the RNAi pathway. In vivo, VTP treatment protected mice against lethal EV-A71 challenge, with detectable vsiRNAs. Our findings provide evidence for the functional relevance of RNAi in mammalian immunity and present a therapeutic strategy for infectious disease., Competing Interests: Declaration of interests Wuhan Institute of Virology and Fudan University on behalf of the authors X.Z., L.L., Y.Fang, S.J., Z.L., and Y.Q. have filed a patent application for the use of VTPs (international application PCT/CN2019/072455, Chinese patent application 201980005805.6, U.S. patent application 16/963,196, Japan patent application 2020-560535, and Australian patent application 2019208788). All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Enteroviruses (EV) have been linked to lymphocytic meningitis and exanthems, but they may also be involved in acute gastroenteritis (AGE), a condition whose aetiological agent often remains unidentified. In this work 1214 samples from individuals with AGE were studied with the aim of establishing the incidence of EV. The samples were collected between September and December in three different years and subjected to real-time genomic amplification in order to determine the viral load (VL). Of the 1214 samples studied, infection by a single virus was found in 328 cases (27%) and coinfection in 69 (5.7%). While adenoviruses (AdV) were the most frequent (14.8% of total), EV were present in 126 (10.4%) of the individuals tested. Of the 126 EV-positive samples, this virus was found as a single infection and coinfection in 76 (6.3%) and 50 (4.1%) cases, respectively. VL for EV was 5.58±1.51 log copies/ml (range 3.73-9.69) in the former and 6.27±1.75 (range 3.73-10.5) (p=0.02) in the latter. EV were identified in 97 children under 5 (16.9%) and in 29 (4.5%) patients over 5. Patients less than 5 years showed a higher VL that those more than 5 years age [6.08±1.57 (range 3.82-9.69) vs. 5.07±1.53 (range 3.73-10.58); (p=0.002)]. There was a high incidence of EV in AGE patients, and they were more frequent in those under 5, where they were found to replicate more efficiently. These results therefore indicate that testing for EV should be included in the diagnosis of AGE., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Porcine sapelovirus (PSV) infections have been associated with a wide spectrum of symptoms, ranging from asymptomatic infection to clinical signs including diarrhoea, pneumonia, reproductive disorders, and polioencephalomyelitis. Although it has a global distribution, there have been relatively few studies on PSV in domestic animals. We isolated a PSV strain, SHCM2019, from faecal specimens from swine, using PK-15 cells. To investigate its molecular characteristics and pathogenicity, the genomic sequence of strain SHCM2019 was analysed, and clinical manifestations and pathological changes occurring after inoculation of neonatal piglets were observed. The virus isolated using PK-15 cells was identified as PSV using RT-PCR, transmission electron microscopy (TEM), and immunofluorescence assay (IFA). Sequencing results showed that the full-length genome of the SHCM2019 strain was 7,567 nucleotides (nt) in length, including a 27-nucleotide poly(A) tail. Phylogenetic analysis demonstrated that this virus was a PSV isolate belonging to the Chinese strain cluster. Recombination analysis indicated that there might be a recombination breakpoint upstream of the 3D region of the genome. Pathogenicity experiments demonstrated that the virus isolate could cause diarrhoea and pneumonia in piglets. In breif, a recombinant PSV strain, SHCM2019, was isolated and shown to be pathogenic. Our results may provide a reference for future research on the pathogenic mechanism and evolutionary characteristics of PSV., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Equine parvovirus-hepatitis (EqPV-H) is a newly identified etiologic agent of Theiler's disease (TD). We present a case of EqPV-H-related fulminant hepatitis in a 14-year-old thoroughbred mare in Korea. The mare had acute hepatopathy and gastrointestinal symptoms, with abnormal liver-related blood parameters. The horse was born in the USA and imported to Korea in 2017, with no history of administration of equine biological products after entry into Korea. The horse was diagnosed with EqPV-H-associated hepatitis after abdominal ultrasonography, laparotomy, and nested polymerase chain reaction (PCR) and in situ hybridization (ISH) assays. The serum, nasal swab, oral swab, and liver biopsy were positive for EqPV-H according to the PCR assay. Genetic analysis of the partial NS1 gene of EqPV-H showed a unique nucleotide substitution, distinct from that in previously deposited strains. EqPV-H DNA was found not only in hepatocytes but also in bile duct epithelium and Kupffer cells, particularly via ISH. To the best of our knowledge, this is the first case of EqPV-H-associated TD in Asia, providing the first clinical evidence for viral shedding from the mouth and nose, and identification of EqPV-H in the liver. This study contributes to a better understanding of the pathological features of EqPV-H-associated TD.

We used wastewater-based epidemiology and amplicon-based long-read high-throughput sequencing for surveillance of enteroviruses (EVs) in Maricopa County, Arizona, Southwest United States. We collected 48 samples from 13 sites in three municipalities between 18 June and 1 October 2020, and filtered (175 mL each; 0.45 µm pore size) and extracted RNA from the filter-trapped solids. The RNA was converted to cDNA and processed through two workflows (Sanger sequencing (SSW) and long-read Illumina sequencing (LRISW)) each including a nested polymerase chain reaction (nPCR) assay. We subjected the ~350 bp amplicon from SSW to Sanger sequencing and the ~1900-2400 bp amplicon from LRISW to Illumina sequencing. We identified EV contigs from 11 of the 13 sites and 41.67% (20/48) of screened samples. Using the LRISW, we detected nine EV genotypes from three species (Enterovirus A (CVA4, EV-A76, EV-A90), Enterovirus B (E14) and Enterovirus C (CVA1, CVA11, CVA13, CVA19 and CVA24)) with Enterovirus C representing approximately 90% of the variants. However, the SSW only detected the five Enterovirus C types. Similarity and phylogenetic analysis showed that multiple Enterovirus C lineages were circulating, co-infecting and recombining in the population during the season despite the SARS-CoV-2 pandemic and the non-pharmaceutical public health measures taken to curb transmission.

Objective: The association between enterovirus infection and type 1 diabetes (T1D) is controversial, and this meta-analysis aimed to explore the correlation., Methods: PubMed, Embase, Web of Science, and Cochrane Database were searched from inception to April 2020. Studies were included if they could provide sufficient information to calculate odds ratios and 95% confidence intervals. All analyses were performed using STATA 15.1., Results: Thirty-eight studies, encompassing 5921 subjects (2841 T1D patients and 3080 controls), were included. The pooled analysis showed that enterovirus infection was associated with T1D ( P < 0.001). Enterovirus infection was correlated with T1D in the European ( P < 0.001), African ( P = 0.002), Asian ( P = 0.001), Australian ( P = 0.011), and Latin American ( P = 0.002) populations, but no conclusion could be reached for North America. The association between enterovirus infection and T1D was detected in blood and tissue samples (both P < 0.001); no association was found in stool samples., Conclusion: Our findings suggest that enterovirus infection is associated with T1D., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Ye, Chen, Xu, Zhao, Wang and Lan.)

Rhinoviruses (RVs) are the main cause of recurrent infections with rather mild symptoms characteristic of the common cold. Nevertheless, RVs give rise to enormous numbers of absences from work and school and may become life-threatening in particular settings. Vaccination is jeopardised by the large number of serotypes eliciting only poorly cross-neutralising antibodies. Conversely, antivirals developed over the years failed FDA approval because of a low efficacy and/or side effects. RV species A, B, and C are now included in the fifteen species of the genus Enteroviruses based upon the high similarity of their genome sequences. As a result of their comparably low pathogenicity, RVs have become a handy model for other, more dangerous members of this genus, e.g., poliovirus and enterovirus 71. We provide a short overview of viral proteins that are considered potential drug targets and their corresponding drug candidates. We briefly mention more recently identified cellular enzymes whose inhibition impacts on RVs and comment novel approaches to interfere with infection via aggregation, virus trapping, or preventing viral access to the cell receptor. Finally, we devote a large part of this article to adding the viral RNA genome to the list of potential drug targets by dwelling on its structure, folding, and the still debated way of its exit from the capsid. Finally, we discuss the recent finding that G-quadruplex stabilising compounds impact on RNA egress possibly via obfuscating the unravelling of stable secondary structural elements.

Picornaviruses are a diverse and major cause of human disease, and their genomes replicate with intracellular membranes. The functionality of these replication organelles depends on the activities of both viral nonstructural proteins and co-opted host proteins. The mechanism by which viral-host interactions generate viral replication organelles and regulate viral RNA synthesis is unclear. To elucidate this mechanism, enterovirus A71 (EV-A71) was used here as a virus model to investigate how these replication organelles are formed and to identify the cellular components that are critical in this process. An immunoprecipitation assay was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify 172 cellular proteins and four viral proteins associating with viral 3A protein. Secretory carrier membrane protein 3 (SCAMP3) was one of the host proteins we selected for further investigation. Here, we demonstrate by immunoprecipitation assay that SCAMP3 associates with 3A protein and colocalizes with 3A protein during virus infection. SCAMP3 knockdown or knockout in infected cells decreases synthesis of EV-A71 viral RNA, viral proteins, and viral growth. Furthermore, the viral 3A protein associates with SCAMP3 and phosphatidylinositol-4-kinase type III β (PI4KIIIβ) as shown by immunoprecipitation assay and colocalizes to the replication complex. Upon infection of cells with a SCAMP3 knockout construct, PI4KIIIβ and phosphatidylinositol-4-phosphate (PI4P) colocalization with EV-A71 3A protein decreases; viral RNA synthesis also decreases. SCAMP3 is also involved in the extracellular signal-regulated kinase (ERK) signaling pathway to regulate viral replication. The 3A and SCAMP3 interaction is also important for the replication of coxsackievirus B3 (CVB3). SCAMP3 also associates with 3A protein of CVB3 and enhances viral replication but does not regulate dengue virus 2 (DENV2) replication. Taken together, the results suggest that enterovirus 3A protein, SCAMP3, PI4KIIIβ, and PI4P form a replication complex and positively regulate enterovirus replication. IMPORTANCE Virus-host interaction plays an important role in viral replication. 3A protein of enterovirus A71 (EV-A71) recruits other viral and host factors to form a replication complex, which is important for viral replication. In this investigation, we utilized immunoprecipitation combined with proteomics approaches to identify 3A-interacting factors. Our results demonstrate that secretory carrier membrane protein 3 (SCAMP3) is a novel host factor that associates with enterovirus 3A protein, phosphatidylinositol-4-kinase type III β (PI4KIIIβ), and phosphatidylinositol-4-phosphate (PI4P) to form a replication complex and positively regulates viral replication. SCAMP3 is also involved in the extracellular signal-regulated kinase (ERK) signaling pathway to regulate viral replication.

Enterovirus D68 (EV-D68), as one of the major pathogens of paediatric respiratory disease, has been widely spread in the population in recent years. As the basis of virus antigenicity, antigenic epitopes are essential to monitoring the transformation of virus antigenicity. However, there is a lack of systematic studies on the antigenic epitopes of EV-D68. In this study, a bioinformatics-based prediction algorithm for human enteroviruses was used to predict the conformational epitopes of EV-D68. The prediction results showed that the conformational epitopes of EV-D68 were clustered into three sites: site 1, site 2, and site 3. Site 1 was located in the "north rim" region of the canyon near the fivefold axis; site 2 was located in the "puff" region near the twofold axis; and site 3 consisted of two parts, one in the "knob" region on the south rim of the canyon and the other in the threefold axis region. The predicted epitopes overlapped highly with the binding regions of four reported monoclonal antibodies (mAbs), indicating that the predictions were highly reliable. Phylogenetic analysis showed that amino acid mutations in the epitopes of the VP1 BC loop, DE loop, C-terminus, and VP2 EF loop played a crucial role in the evolutionary divergence of EV-D68 clades/subclades and epidemics. This finding indicated that the VP1 BC loop, DE loop, C-terminus, and VP2 EF loop were the most important epitopes of EV-D68. Research on the epitopes of EV-D68 will contribute to outbreak surveillance and to the development of diagnostic reagents and recombinant vaccines., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Outbreaks of hand, foot, and mouth disease caused by enterovirus-A71 (EV-A71) can result in many deaths, due to central nervous system complications. Outbreaks with many fatalities have occurred sporadically in the Asia-Pacific region and have become a serious public health concern. It is hypothesized that virulent mutations in the EV-A71 genome cause these occasional outbreaks. Analysis of EV-A71 neurovirulence determinants is important, but there are no virulence determinants that are widely accepted among researchers. This is because most studies have been done in artificially infected mouse models and because EV-A71 mutates very quickly to adapt to the artificial host environment. Although EV-A71 uses multiple receptors for infection, it is clear that adaptation-related mutations alter the binding specificity of the receptors and allow the virus to adopt the best entry route for each environment. Such mutations have confused interpretations of virulence in animal models. This article will discuss how environment-adapted mutations in EV-A71 occur, how they affect virulence, and how such mutations can be avoided. We also discuss future perspectives for EV-A71 virulence research.

Enterovirus D68 (EV-D68) has emerged as an agent of epidemic respiratory illness and acute flaccid myelitis in the paediatric population but data are lacking in adult patients. We performed a 4.5-year single-centre retrospective study of all patients who tested positive for EV-D68 and analysed full-length EV-D68 genomes of the predominant clades B3 and D1. Between 1 June 2014, and 31 December 2018, 73 of the 11,365 patients investigated for respiratory pathogens tested positive for EV-D68, of whom 20 (27%) were adults (median age 53.7 years [IQR 34.0-65.7]) and 53 (73%) were children (median age 1.9 years [IQR 0.2-4.0]). The proportion of adults increased from 12% in 2014 to 48% in 2018 ( p = 0.01). All adults had an underlying comorbidity factor, including chronic lung disease in 12 (60%), diabetes mellitus in six (30%), and chronic heart disease in five (25%). Clade D1 infected a higher proportion of adults than clades B3 and B2 ( p = 0.001). Clade D1 was more divergent than clade B3: 5 of 19 amino acid changes in the capsid proteins were located in putative antigenic sites. Adult patients with underlying conditions are more likely to present with severe complications associated with EV-D68, notably the emergent clade D1.

Aquaporin-4 (AQP4) is the key water channel protein that regulates brain water homeostasis. Polarized expression of AQP4 on the astroglial endfeet facilitates its role in bi-directional brain water flux control. In the current study, we found that enterovirus 71 (EV71) infection induced depolarization of AQP4 in mouse brain, and demonstrated that β-dystroglycan (β-DG), the key component of dystrophin glycoprotein complex (DGC) that anchors AQP4 to the astroglial endfeet, was degraded upon infection. Elevated activity or expression of matrix metalloproteinase 9 (MMP9) upon infection was found in both mouse brains and patient cerebrospinal fluid (CSF) samples. Inhibiting MMP9 activity by SB-3CT rescued the decay of β-DG and reduced the depolarization of AQP4. Brain edema induced by viral infection was also ameliorated by SB-3CT treatment in mice., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: To test the hypothesis that enterovirus meningitis (EM) is a frequent and self-limiting condition, the epidemiology of EM in adults was examined., Methods: Using a prospective, nationwide, population-based database, all adults with EM confirmed by PCR of the CSF from 2015 to 2019 were included. Unfavorable outcome was defined as Glasgow Outcome Scale scores of 1-4 at discharge. Modified Poisson regression was used to compute adjusted relative risks (RRs)., Results: A total of 419 cases of EM in 418 adults (46% female, median age 31 years [interquartile range (IQR) 27-35]) yielded an incidence of 1.80/100,000/year. Admission diagnoses included CNS infection 247/397 (62%), other neurologic conditions 89/397 (22%), and cerebrovascular diseases 33/397 (8%). Genotype was available for 271 cases, of which echovirus 30 accounted for 155 (57%). Patients presented with headache 412/415 (99%), history of fever 303/372 (81%), photophobia 292/379 (77%), and neck stiffness 159/407 (39%). Fever (≥38.0°C) was observed in 192/399 (48%) at admission. The median CSF leukocyte count was 130 10 6 /L (range 0-2,100) with polymorphonuclear predominance (>50%) in 110/396 (28%). Cranial imaging preceded lumbar puncture in 127/417 (30%) and was associated with non-CNS infection admission diagnoses and delayed lumbar puncture (median 4.8 hours [IQR 3.4-7.9] vs 1.5 [IQR 0.8-2.8], p < 0.001). Unfavorable outcome occurred in 99/419 (24%) at discharge; more often in female patients (RR 2.30 [1.58-3.33]) and less frequent in echovirus 30 (RR 0.67 [0.46-1.00]) in adjusted analyses. Outcome remained unfavorable in 22/379 (6%) after 6 months., Conclusions: EM is common among young, healthy adults. Although the long-term prognosis remains reassuring, a substantial proportion have moderate disability at discharge, especially female patients., (© 2021 American Academy of Neurology.)

The generation of a human pancreatic beta cell line which reproduces the responses seen in primary beta cells, but is amenable to propagation in culture, has long been an important goal in diabetes research. This is particularly true for studies focussing on the role of enteroviral infection as a potential cause of beta-cell autoimmunity in type 1 diabetes. In the present work we made use of a clonal beta cell line (1.1B4) available from the European Collection of Authenticated Cell Cultures, which had been generated by the fusion of primary human beta-cells with a pancreatic ductal carcinoma cell, PANC-1. Our goal was to study the factors allowing the development and persistence of a chronic enteroviral infection in human beta-cells. Since PANC-1 cells have been reported to support persistent enteroviral infection, the hybrid 1.1B4 cells appeared to offer an ideal vehicle for our studies. In support of this, infection of the cells with a Coxsackie virus isolated originally from the pancreas of a child with type 1 diabetes, CVB4.E2, at a low multiplicity of infection, resulted in the development of a state of persistent infection. Investigation of the molecular mechanisms suggested that this response was facilitated by a number of unexpected outcomes including an apparent failure of the cells to up-regulate certain anti-viral response gene products in response to interferons. However, more detailed exploration revealed that this lack of response was restricted to molecular targets that were either activated by, or detected with, human-selective reagents. By contrast, and to our surprise, the cells were much more responsive to rodent-selective reagents. Using multiple approaches, we then established that populations of 1.1B4 cells are not homogeneous but that they contain a mixture of rodent and human cells. This was true both of our own cell stocks and those held by the European Collection of Authenticated Cell Cultures. In view of this unexpected finding, we developed a strategy to harvest, isolate and expand single cell clones from the heterogeneous population, which allowed us to establish colonies of 1.1B4 cells that were uniquely human (h1.1.B4). However, extensive analysis of the gene expression profiles, immunoreactive insulin content, regulated secretory pathways and the electrophysiological properties of these cells demonstrated that they did not retain the principal characteristics expected of human beta cells. Our data suggest that stocks of 1.1B4 cells should be evaluated carefully prior to their use as a model human beta-cell since they may not retain the phenotype expected of human beta-cells., (© 2021. The Author(s).)

Enteroviruses (EVs) are RNA viruses that can cause many clinical syndromes including acute flaccid paralysis (AFP). Within the global polio laboratory network, EVs are categorized either as polioviruses or non-polio enteroviruses (NPEVs). Specific NPEVs have been described in polio-like residual paralytic events in AFP patients. Retrospective analysis of 112 NPEV isolates from AFP patients was performed and thirty one NPEV types were identified of which 91% were Enterovirus B and 9% were Enterovirus A species. The NPEVs were distributed across the country with most patients in the eastern region (41/89; 46.1%). The highest proportion of patients were children less than 5 years (77/89; 86.5%) and male patients were more common (54/89; 60.7%). Echovirus 11 (11/89; 12.4%) was frequently observed and phylogenetic analysis of these sequences revealed high diversity. Coxsackievirus B5 (CV-B5), CV-B6, E21, and EV-B69 were only seen in patients with residual paralysis. Analyses of the EV-A71 sequence indicated a unique genogroup., (© 2021 The Authors. Journal of Medical Virology Published by Wiley Periodicals LLC.)

As one of the principal etiological agents of hand, foot, and mouth disease (HFMD), enterovirus 71 (EV71) is associated with severe neurological complications or fatal diseases, while without effective medications thus far. Here we applied dually activated Michael acceptor to develop a series of reversible covalent compounds for EV71 3C protease (3C pro ), a promising antiviral drug target that plays an essential role during viral replication by cleaving the precursor polyprotein, inhibiting host protein synthesis, and evading innate immunity. Among them, cyanoacrylate and Boc-protected cyanoarylamide derivatives (SLQ-4 and SLQ-5) showed effective antiviral activity against EV71. The two inhibitors exhibited broad antiviral effects, acting on RD, 293T, and Vero cell lines, as well as on EV71 A, B, C, CVA16, and CVB3 viral strains. We further determined the binding pockets between the two inhibitors and 3C pro based on docking studies. These results, together with our previous studies, provide evidence to elucidate the mechanism of action of these two reversible covalent inhibitors and contribute to the development of clinically effective medicines to treat EV71 infections., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Acute flaccid myelitis (AFM) is an incompletely understood neurologic disorder occurring in epidemic fashion causing weakness ranging from mild paresis to devastating paralysis in children and some adults. This article reviews the case definition of AFM as well as its epidemiology and association with enteroviral infection. The clinical presentation, diagnostic investigation with particular attention to electrodiagnostics, acute management, and surgical options are described. Clinical outcomes and considerations for acute and long-term rehabilitation management are discussed extensively based on review of current literature, highlighting avenues for further study., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Enteroviruses A71 (EVs-A71) are known to cause serious neurological infections, especially in the pediatric population. We report here eight cases of EV-A71 infection diagnosed in Marseille over the past 2 years (seven cases in 2019 and one case in 2020). Only children under 5 years of age were affected, including one case of acute flaccid paralysis. Viral RNA was detected by RT-PCR in peripheral samples for all cases (feces and upper respiratory samples). Phylogenetic analyses based on VP1 and 2C3C coding regions revealed that all these cases of EV-A71 infection were caused by viruses belonging to the subgenogroup C1 that currently circulates in Europe and that these viruses are genetically closed to other EVs-A71 recently detected in European countries. These data therefore reinforce the usefulness of the enterovirus surveillance network and the need for systematic screening for EV-A71 in case of an enteroviral infection. This study therefore suggests that the systematic screening for EV-A71 in case of enteroviral infection could provide additional data for enterovirus surveillance networks., (© 2021 Wiley Periodicals LLC.)

MicroRNAs (miRNAs) are crucial in the process of host-pathogen interaction. In this study, we established a screening system for miRNAs of target genes to detect the effect of miRNAs on Enterovirus 71 (EV71) replication in rhabdomyosarcoma (RD) cells. A 3'-untranslated region (UTR) dual-luciferase assay was performed to confirm putative miRNA targets in EV71 genome. Firstly, 13 fragments of EV71 genome were inserted into the vector pMIR, and luciferase activities were analyzed to identify the putative miRNAs of target genes. The expression of the reporter protein was significantly downregulated in cells transfected with the vector containing gene VP3. Then we screened for miRNAs that might target to VP3 through online analysis software. In addition, Western blot, real-time PCR, virus titration, and morphological changes were considered to examine the effects of miRNAs on virus replication. The results suggested that miR-18a and miR-452 repress the reproduction of EV71 virus by binding to VP3. Moreover, EV71 infection also affected the expression of endogenous miR-18a and miR-452. In addition, no significant cytotoxic effects were observed. The results from this study suggest that the intracellular miRNAs may play vital roles in the host-virus interaction.

Enterovirus A71 (EV-A71 or EV-71) is an RNA virus that causes hand, foot and mouse disease in children. The N6-methyladenosine (m6A) of RNA is a common RNA modification involved in various biological events. METTL3 is an m6A methyltransferase that regulates EV-71 replication. EV-71 infection induces autophagy, which also promotes EV-71 replication. In this study, we explored the role of METTL3 in EV-71 infection-induced autophagy. We constructed lentivirus expressing METTL3-specific shRNA and knocked down the endogenous METTL3 in mouse Schwann cells. We infected normal Schwann cells and METTL3 knockdown Schwann cells and compared the viral titer, expression of autophagy-related proteins and apoptosis-related protein. Transduction of lentivirus expressing METTL3 shRNA significantly decreased the endogenous METTL3. Knocking down METTL3 decreased the viral titer of EV-71 after infection. Knocking down METTL3 prevented EV-71-induced cell death and suppressed EV-71-induced expression of Bax while rescuing Bcl-2 expression after EV-71 infection. Knocking down METTL3 inhibited EV-71-induced expression of Atg5, Atg7 and LC3 II. Knocking down METTL3 inhibited EV-71-induced apoptosis and autophagy. In summary, our study describes the relationship of METTL3 and autophagy during EV-71 infection., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

RNA viruses exist as genetically heterogeneous populations due to high mutation rates, and many of these mutations reduce fitness and/or replication speed. However, it is unknown whether mutations can increase replication speed of a virus already well adapted to replication in cultured cells. By sequentially passaging coxsackievirus B3 in cultured cells and collecting the very earliest progeny, we selected for increased replication speed. We found that a single mutation in a viral capsid protein, VP1-F106L, was sufficient for the fast-replication phenotype. Characterization of this mutant revealed quicker genome release during entry compared to wild-type virus, highlighting a previously unappreciated infection barrier. However, this mutation also reduced capsid stability in vitro and reduced replication and pathogenesis in mice. These results reveal a tradeoff between overall replication speed and fitness. Importantly, this approach-selecting for the earliest viral progeny-could be applied to a variety of viral systems and has the potential to reveal unanticipated inefficiencies in viral replication cycles., Competing Interests: The authors declare no competing interest.

Enterovirus 71 (EV71) is a positive single-stranded RNA virus from the enterovirus genus of the Picornaviridae family. Most young children infected with EV71 develop mild symptoms of hand, foot and mouth disease, but some develop severe symptoms with neurological involvement. Limb paralysis from EV71 infection is presumed to arise mainly from dysfunction of motor neurons in the spinal cord. However, EV71 also targets and damages skeletal muscle, which may also contribute to the debilitating symptoms. In this study, we have delineated the impacts of EV71 infection on skeletal muscle using a mouse model. Mouse pups infected with EV71 developed limb paralysis, starting at day 3 post-infection and peaking at day 5-7 post-infection. At later times, mice recovered gradually but not completely. Notably, severe disease was associated with high levels of myositis accompanied by muscle calcification and persistent motor end plate abnormalities. Interestingly, macrophages exhibited a dynamic change in phenotype, with inflammatory macrophages (CD45 + CD11b + Ly6C hi ) appearing in the early stage of infection and anti-inflammatory/restorative macrophages (CD45 + CD11b + Ly6C low/- ) appearing in the late stage. The presence of inflammatory macrophages was associated with severe inflammation, while the restorative macrophages were associated with recovery. Altogether, we have demonstrated that EV71 infection causes myositis, muscle calcification and structural defects in motor end plates. Subsequent muscle regeneration is associated with a dynamic change in macrophage phenotype., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Lin, Kuo, Chuang, Wang and Liao.)

The emergence of SARS-CoV-2 and subsequent COVID-19 pandemic highlights the morbidity and potential disease severity caused by respiratory viruses. To elucidate pathogen prevalence, etiology of coinfections and URIs from symptomatic adult Emergency department patients in a pre-SARS-CoV-2 environment, we evaluated specimens from four geographically diverse Emergency departments in the United States from 2013-2014 utilizing ePlex RP RUO cartridges (Genmark Diagnostics). The overall positivity was 30.1% (241/799), with 6.6% (16/241) coinfections. Noninfluenza pathogens from most to least common were rhinovirus/enterovirus, coronavirus, human metapneumovirus and RSV, respectively. Broad differences in disease prevalence and pathogen distributions were observed across geographic regions; the site with the highest detection rate (for both mono and coinfections) demonstrated the greatest pathogen diversity. A variety of respiratory pathogens and geographic variations in disease prevalence and copathogen type were observed. Further research is required to evaluate the clinical relevance of these findings, especially considering the SARS-CoV-2 pandemic and related questions regarding SARS-CoV-2 disease severity and the presence of co-infections., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Human enteroviruses are the most prevalent causes of aseptic meningitis worldwide. However, despite such predominancy, defining the enteroviral etiology of aseptic meningitis remains a diagnostic dilemma for the clinician in Iran. Therefore, this study was conducted to characterize the prevalence and clinical significance of enteroviral aseptic meningitis as well as the predominant enterovirus serotypes among patients with aseptic meningitis in the South of Iran.Cerebrospinal fluid (CSF) specimens were obtained from 73 patients with aseptic meningitis (52.1% males and 47.9% females), ages ranging from 1 month to 88 years. Following the extraction of nucleic acid, the detection of enteroviruses was performed by RT-PCR, targeting the 5' untranslated region of the genome, and sequencing. Enteroviruses were found in 46.6% of samples (34/73). The most predominant serotype was echovirus 30, followed by coxsackievirus B5 and poliovirus type 1 Sabin strain. The enterovirus infections were more prevalent among female patients (58.8%) and those below 5 years of age (52.9%). Although enterovirus infections were observed throughout the year, the infections were more prevalent during autumn with fever as the predominant clinical symptom. The outcomes revealed that enteroviruses are significant causes of aseptic meningitis in the South of Iran, while suspected cases of aseptic meningitis are usually monitored by bacterial culture and biochemical testing of CSF samples. Therefore, the etiology remains unknown in most cases. Molecular detection of viral pathogens should be included as a common approach in the screening of patients with aseptic meningitis to prevent unnecessary treatment and to improve clinical management.

Human Enteroviruses (hEVs) are responsible for a wide variety of human diseases. During hEVs infection, virions are excreted in human feces and the fecal-oral route is the primary pathway for person-to-person transmission. Sewage surveillance could help in monitoring hEVs circulation and describing their diversity in a specific population. In this study, sewage samples collected in Buenos Aires Metropolitan Area (Argentina) were retrospectively studied through an amplicon-deep sequencing approach and phylogenetic analyses to characterize hEVs spread. We identified 17 different hEVs types belonging to A, B, and C species. To the best of our knowledge, this is the first report in Buenos Aires for 7 identified hEV-C types. Phylogenetic analyses suggest several introductions of coxsackievirus B4, echovirus 1, and echovirus 9 in the country, along with the national spread reached by some variants. Besides, well-supported monophyletic groups of Argentine, Uruguayan, and Brazilian strains unveiled regional circulation patterns for some variants. These results extend our knowledge about hEVs circulation in Buenos Aires and might exhort authorities to implement more active sewage surveillance in the region.

To investigate the seroepidemiological features of enterovirus D68 (EV-D68) in the healthy population from 2012 to 2017 in Beijing, China. A retrospective cross-sectional investigation was conducted using serum specimens collected from healthy individuals in Beijing from 2012 to 2017. These samples were tested for neutralization antibodies (NtAbs) against EV-D68. The sera from six EV-D68 infected patients in the acute or convalescent phase were used to determine the protection level of NtAbs against EV-D68. The geometric means of the titers (GMT) of EV-D68 NtAbs in 2012 and 2017 were 92.82 and 242.91, respectively; the seroprevalences of EV-D68 were 89.43% and 98.43%, respectively. The GMT reached its peak in the 11 to 15 age group in 2012, while in 16 to 20 age group in 2017. We also observed that EV-D68 NtAbs titers of six sera from the acute phase were all less than equal to  1:64 and that of three sera from the convalescent phase were all more than 1:64. Anti-EV-D68 NtAbs in the population remained low from 2012 to 2016 but increased significantly in 2017. Although most of the EV-D68 infections remain undetected in Beijing, the risk of a large outbreak of EV-D68 exists and should be taken seriously., (© 2020 Wiley Periodicals LLC.)

Rodents are common reservoirs for numerous zoonotic pathogens, but knowledge about diversity of pathogens in rodents is still limited. Here, we investigated the occurrence and genetic diversity of enteric viruses in 51 Norway rats collected in three different countries in Europe. RNA of at least one virus was detected in the intestine of 49 of 51 animals. Astrovirus RNA was detected in 46 animals, mostly of rat astroviruses. Human astrovirus (HAstV-8) RNA was detected in one, rotavirus group A (RVA) RNA was identified in eleven animals. One RVA RNA could be typed as rat G3 type. Rat hepatitis E virus (HEV) RNA was detected in five animals. Two entire genome sequences of ratHEV were determined. Human norovirus RNA was detected in four animals with the genotypes GI.P4-GI.4, GII.P33-GII.1, and GII.P21. In one animal, a replication competent coxsackievirus A20 strain was detected. Additionally, RNA of an enterovirus species A strain was detected in the same animal, albeit in a different tissue. The results show a high detection rate and diversity of enteric viruses in Norway rats in Europe and indicate their significance as vectors for zoonotic transmission of enteric viruses. The detailed role of Norway rats and transmission pathways of enteric viruses needs to be investigated in further studies.

Enteroviruses belong to the genus Enterovirus of the family Picornaviridae and include four human enterovirus groups (EV-A to -D): the epidemic of enteroviruses such as human enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) is a threat to global public health. Enteroviral protein 2C is the most conserved nonstructural protein among all enteroviruses and possesses RNA helicase activity that plays pivotal roles during enteroviral life cycles, which makes 2C an attractive target for developing antienterovirus drugs. In this study, we designed a peptide, named 2CL, based on the structure of EV-A71 2C. This peptide effectively impaired the oligomerization of EV-A71 2C protein and inhibited the RNA helicase activities of 2C proteins encoded by EV-A71 and CVA16, both of which belong to EV-A, and showed potent antiviral efficacy against EV-A71 and CVA16 in cells. Moreover, the 2CL treatment elicited a strong in vivo protective efficacy against lethal EV-A71 challenge. In addition, the antiviral strategy of targeting the 2C helicase activity can be applied to inhibit the replication of EV-B. Either 2CL or B-2CL, the peptide redesigned based on the 2CL-corresponding sequence of EV-Bs, could exert effective antiviral activity against two important EV-Bs, coxsackievirus B3 and echovirus 11. Together, our findings demonstrated that targeting the helicase activity of 2C with a rationally designed peptide is an efficient antiviral strategy against enteroviruses, and 2CL and B-2CL show promising clinical potential to be further developed as broad-spectrum antienterovirus drugs. IMPORTANCE Enteroviruses are a large group of positive-sense single-stranded RNA viruses and include numerous human pathogens, such as enterovirus A71 (EV-A71), coxsackieviruses, and echoviruses. However, no approved EV antiviral drugs are available. Enteroviral 2C is the most conserved nonstructural protein among all enteroviruses and contains the RNA helicase activity critical for the viral life cycle. Herein, according to the structure of EV-A71 2C, we designed a peptide that effectively inhibited the RNA helicase activities of EV-A71- and coxsackievirus A16 (CVA16)-encoded 2C proteins. Moreover, this peptide exerted potent antiviral effects against EV-A71 and CVA16 in cells and elicited therapeutic efficacy against lethal EV-A71 challenge in vivo Furthermore, we demonstrate that the strategy of targeting the 2C helicase activity can be used for other relevant enteroviruses, including coxsackievirus B3 and echovirus 11. In summary, our findings provide compelling evidence that the designed peptides targeting the helicase activity of 2C could be broad-spectrum antivirals for enteroviruses., (Copyright © 2021 American Society for Microbiology.)

Enterovirus D68 (EV-D68) is an emerging pathogen associated with respiratory diseases and/or acute flaccid myelitis. Here, two MAbs, 2H12 and 8F12, raised against EV-D68 virus-like particle (VLP), show distinct preference in binding VLP and virion and in neutralizing different EV-D68 strains. A combination of 2H12 and 8F12 exhibits balanced and potent neutralization effects and confers broader protection in mice than single MAbs when given at onset of symptoms. Cryo-EM structures of EV-D68 virion complexed with 2H12 or 8F12 show that both antibodies bind to the canyon region of the virion, creating steric hindrance for sialic acid receptor binding. Additionally, 2H12 binding can impair virion integrity and trigger premature viral uncoating. We also capture an uncoating intermediate induced by 2H12 binding, not previously described for picornaviruses. Our study elucidates the structural basis and neutralizing mechanisms of the 2H12 and 8F12 MAbs and supports further development of the 2H12/8F12 cocktail as a broad-spectrum therapeutic agent against EV-D68 infections in humans.

Enteroviruses are associated with various diseases accompanied by rare but severe complications. In recent years, outbreaks of enterovirus D68 and enterovirus A71 associated with severe respiratory infections and neurological complications have been reported worldwide. Since information on molecular epidemiology in respiratory samples is still limited, the genetic diversity of enteroviruses was retrospectively analysed over a 4-year period (2013-2016) in respiratory samples from paediatric patients. Partial viral major capsid protein gene (VP1) sequences were determined for genotyping. Enteroviruses were detected in 255 (6.1%) of 4187 specimens. Phylogenetic analyses of 233 (91.4%) strains revealed 25 different genotypes distributed to Enterovirus A (39.1%), Enterovirus B (34.3%), and Enterovirus D (26.6%). The most frequently detected genotypes were enterovirus D68 (26.6%), coxsackievirus A6 (15.9%), and enterovirus A71 (7.3%). Enterovirus D68 detections were associated with lower respiratory tract infections and increased oxygen demand. Meningitis/encephalitis and other neurological symptoms were related to enterovirus A71, while coxsackievirus A6 was associated with upper respiratory diseases. Prematurity turned out as a potential risk factor for increased oxygen demand during enterovirus infections. The detailed analysis of epidemiological and clinical data contributes to the non-polio enterovirus surveillance in Europe and showed high and rapidly changing genetic diversity of circulating enteroviruses, including different enterovirus D68 variants.

Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14-ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA-VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid-RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Abstract: Enteroviruses is a group of positive single-stranded RNA viruses ubiquitous in the environment, which is a causative agent of epidemic diseases in children and infants. But data on neonates are still limited. The present study aimed to describe the clinical characteristics of enterovirus infection in neonates and arise the awareness of this disease to general public.Between March 2018 and September 2019, data from all of the neonates diagnosed with enterovirus infection were collected and analyzed from neonatal intensive care unit of Zhangzhou Hospital in Fujian, China.A total of 23 neonates were enrolled. All of them presented with fever (100%), and some with rashes (39.1%). The incidence of aseptic meningitis was high (91.3%), but only a small proportion (28.6%) presented with cerebrospinal fluid (CSF) leukocytosis. The positive value for nucleic acid detection in CSF was significantly higher than throat swab (91.3% vs 43.5%, P = .007). Five of the infected neonates presented with aseptic meningitis (23.8%) underwent brain magnetic resonance imaging examination and no craniocerebral injuries were found. Subsequent follow-ups were performed in 15 of them (71.4%) and no neurological sequelae was found.Aseptic meningitis is a common type of enterovirus infection in neonates with a benign course. Nucleic acid detection of CSF has an important diagnostic value. Febrile neonates would be suggested to screen for enterovirus infection in addition to complete septic workup. An unnecessary initiation or earlier cessation of antibiotics could be considered in enterovirus infection, but that indications still need further studies to guarantee the safety., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Due to the outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), combined with the risk of polio importation from Ukraine, we evaluated the presence of SARS-CoV-2 and enteroviruses in 25 sewage water samples from Romania, concentrated using the WHO method between January 2020 and January 2021. Surveillance for enteroviruses and SARS-CoV-2 are relevant in the calculation of prevalence estimates as well as early detection of the introduction or disappearance of these viruses. For SARS-CoV-2 detection, we used two immunochromatographic nucleocapsid antigenic tests as well as real-time PCR assays, produced for respiratory samples. The isolation of cell culture lines, in accordance with the WHO recommendations, was carried out for enterovirus detection. Twenty-three of the samples investigated were positive in rapid tests for SARS-CoV-2, while the RNA of SARS-CoV-2, detected with Respiratory 2.1 plus a panel Biofire Film array, was present in eight samples. The Allplex 2019-nCoV assay was used for the validation of the tests. There were three genes detected in one sample, E, RdPR, and N, two genes, E and RdPR, in one sample, two genes, RdPR and N, in four samples, one gene, RdPR, in five samples and one gene, N, in one sample. Eight samples were positive for non-polio enteroviruses, and no poliovirus strains were isolated. This study suggests the presence of SARS-CoV-2 and enteroviruses in Romanian sewage water in 2020. As such, our results indicate that a rapid, more specific test should be developed especially for the detection of SARS-CoV-2 in sewage water.