VP1 is the primary determinant of neuropathogenesis in a mouse model of enterovirus D68 acute flaccid myelitis.

Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory and neurologic disease [acute flaccid myelitis (AFM)]. Intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with US/IL/14-18952 (IL52), a clinical isolate from the 2014 EV-D68 epidemic, results in many of the pathogenic features of human AFM, including viral infection of the spinal cord, death of motor neurons, and resultant progressive paralysis. In distinction, CA/14-4231 (CA4231), another clinical isolate from the 2014 EV-D68 outbreak, does not cause paralysis in mice, does not grow in the spinal cord, and does not cause motor neuron loss following IM injection. A panel of chimeric viruses containing sequences from IL52 and CA4231 was used to demonstrate that VP1 is the main determinant of EV-D68 neurovirulence following IM injection of neonatal SW mice. VP1 contains four amino acid differences between IL52 and CA4231. Mutations resulting in substituting these four amino acids (CA4231 residues into the IL52 polyprotein) completely abolished neurovirulence. Conversely, mutations resulting in substituting VP1 IL52 amino acid residues into the CA4231 polyprotein created a virus that induced paralysis to the same degree as IL52. Neurovirulence following infection of neonatal SW mice with parental and chimeric viruses was associated with viral growth in the spinal cord. IMPORTANCE: Emerging viruses allow us to investigate mutations leading to increased disease severity. Enterovirus D68 (EV-D68), once the cause of rare cases of respiratory illness, recently acquired the ability to cause severe respiratory and neurologic disease. Chimeric viruses were used to demonstrate that viral structural protein VP1 determines growth in the spinal cord, motor neuron loss, and paralysis following intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with EV-D68. These results have relevance for predicting the clinical outcome of future EV-D68 epidemics as well as targeting retrograde transport as a potential strategy for treating virus-induced neurologic disease.

Clinical and molecular epidemiology of enterovirus D68 from 2013 to 2020 in Shanghai.

Enterovirus D68 (EV-D68) is an emerging pathogen that has caused outbreaks of severe respiratory disease worldwide, especially in children. We aim to investigate the prevalence and genetic characteristics of EV-D68 in children from Shanghai. Nasopharyngeal swab or bronchoalveolar lavage fluid samples collected from children hospitalized with community-acquired pneumonia were screened for EV-D68. Nine of 3997 samples were EV-D68-positive. Seven of nine positive samples were sequenced and submitted to GenBank. Based on partial polyprotein gene (3D) or complete sequence analysis, we found the seven strains belong to different clades and subclades, including three D1 (detected in 2013 and 2014), one D2 (2013), one D3 (2019), and two B3 (2014 and 2018). Overall, we show different clades and subclades of EV-D68 spread with low positive rates (0.2%) among children in Shanghai between 2013 and 2020. Amino acid mutations were found in the epitopes of the VP1 BC and DE loops and C-terminus; similarity analysis provided evidence for recombination as an important mechanism of genomic diversification. Both single nucleotide mutations and recombination play a role in evolution of EV-D68. Genetic instability within these clinical strains may indicate large outbreaks could occur following cumulative mutations.

SARS-CoV-2 and oncolytic EV-D68-encoded proteases differentially regulate pyroptosis.

Pyroptosis, a pro-inflammatory programmed cell death, has been implicated in the pathogenesis of coronavirus disease 2019 and other viral diseases. Gasdermin family proteins (GSDMs), including GSDMD and GSDME, are key regulators of pyroptotic cell death. However, the mechanisms by which virus infection modulates pyroptosis remain unclear. Here, we employed a mCherry-GSDMD fluorescent reporter assay to screen for viral proteins that impede the localization and function of GSDMD in living cells. Our data indicated that the main protease NSP5 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) blocked GSDMD-mediated pyroptosis via cleaving residues Q29 and Q193 of GSDMD. While another SARS-CoV-2 protease, NSP3, cleaved GSDME at residue G370 but activated GSDME-mediated pyroptosis. Interestingly, respiratory enterovirus EV-D68-encoded proteases 3C and 2A also exhibit similar differential regulation on the functions of GSDMs by inactivating GSDMD but initiating GSDME-mediated pyroptosis. EV-D68 infection exerted oncolytic effects on human cancer cells by inducing pyroptotic cell death. Our findings provide insights into how respiratory viruses manipulate host cell pyroptosis and suggest potential targets for antiviral therapy as well as cancer treatment.IMPORTANCEPyroptosis plays a crucial role in the pathogenesis of coronavirus disease 2019, and comprehending its function may facilitate the development of novel therapeutic strategies. This study aims to explore how viral-encoded proteases modulate pyroptosis. We investigated the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory enterovirus D68 (EV-D68) proteases on host cell pyroptosis. We found that SARS-CoV-2-encoded proteases NSP5 and NSP3 inactivate gasdermin D (GSDMD) but initiate gasdermin E (GSDME)-mediated pyroptosis, respectively. We also discovered that another respiratory virus EV-D68 encodes two distinct proteases 2A and 3C that selectively trigger GSDME-mediated pyroptosis while suppressing the function of GSDMD. Based on these findings, we further noted that EV-D68 infection triggers pyroptosis and produces oncolytic effects in human carcinoma cells. Our study provides new insights into the molecular mechanisms underlying virus-modulated pyroptosis and identifies potential targets for the development of antiviral and cancer therapeutics.

Clinical and molecular epidemiologic features of enterovirus D68 infection in children with acute lower respiratory tract infection in China.

Acute flaccid paralysis (AFP) associated with enterovirus D68 (EV-D68) infection has attracted much attention since an outbreak in the USA in 2014. Notably, EV-D68 was detected in a child with AFP for the first time in China in 2018. In a multicentre study from May 2017 to December 2019, we monitored EV-D68 infections in hospitalized children with acute lower respiratory tract infection (ALRTI) in China. Out of 3,071 samples collected from patients with ALRTI, ten were positive for EV-D68. All patients presented with mild diseases with no neurological symptoms or signs. Phylogenetic analysis based on the VP1 gene showed that all EV-D68 sequences obtained in this study belonged to subclade B3 and were close to sequences of EV-D68 strains obtained from patients with AFP in the USA. Four EV-D68 strains were isolated, and their complete genome sequences were determined. These sequences did not show any evidence of recombination events. To assess their neurotropism, the isolates were used to infect the "neuronal-like" cell line SH-SY5Y, and resulted in a cytopathic effect. We further analysed the structure and sites that may be associated with neurovirulence, including the stem-loop structure in the untranslated region (3'UTR) and identified amino acid substitutions (M291T, V341A, T860N, D927N, S1108G, and R2005K) in the coding region and specific nucleotides (127T, 262C, and 339T) in the 5' UTR. In conclusion, EV-D68 infection was detected in a small number of children with ALRTI in China from 2017 to 2019. Disease symptoms in these children were relatively mild with no neurological complications, and all EV-D68 sequences belonged to subclade B3.

ADAR1p110 promotes Enterovirus D68 replication through its deaminase domain and inhibition of PKR pathway.

BACKGROUND: Severe respiratory and neurological diseases caused by human enterovirus D68 (EV-D68) pose a serious threat to public health, and there are currently no effective drugs and vaccines. Adenosine deaminase acting on RNA1 (ADAR1) has diverse biological functions in various viral infections, but its role in EV-D68 infections remains undetermined. METHODS: Rhabdomyosarcoma (RD) and human embryonic kidney 293 T (293 T) cells, and HeLa cells were used to evaluate the expression level of ADAR1 upon EV-D68 (Fermon strain) and human parainfluenza virus type 3 (HPIV3; NIH47885) infection, respectively. Knockdown through silencing RNA (siRNA) and overexpression of either ADAR1p110 or ADAR1p150 in cells were used to determine the function of the two proteins after viral infection. ADAR1p110 double-stranded RNA binding domains (dsRBDs) deletion mutation was generated using a seamless clone kit. The expression of ADAR1, EV-D68 VP1, and HPIV3 hemagglutinin-neuraminidase (HN) proteins was identified using western blotting. The median tissue culture infectious dose (TCID50) was applied to detect viral titers. The transcription level of EV-D68 mRNA was analyzed using reverse transcription-quantitative PCR (RT-qPCR) and the viral 5'-untranslated region (5'-UTR)-mediated translation was analyzed using a dual luciferase reporter system. CONCLUSION: We found that the transcription and expression of ADAR1 was inhibited upon EV-D68 infection. RNA interference of endogenous ADAR1 decreased VP1 protein expression and viral titers, while overexpression of ADAR1p110, but not ADAR1p150, facilitated viral replication. Immunofluorescence assays showed that ADAR1p110 migrated from the nucleus to the cytoplasm after EV-D68 infection. Further, ADAR1p110 lost its pro-viral ability after mutations of the active sites in the deaminase domain, and 5'-UTR sequencing of the viral genome revealed that ADAR1p110 likely plays a role in EV-D68 RNA editing. In addition, after ADAR1 knockdown, the levels of both phosphorylated double-stranded RNA dependent protein kinase (p-PKR) and phosphorylated eukaryotic initiation factor 2α (p-eIF2α) increased. Attenuated translation activity of the viral genome 5'-UTR was also observed in the dual-luciferase reporter assay. Lastly, the deletion of ADAR1p110 dsRBDs increased the level of p-PKR, which correlated with a decreased VP1 expression, indicating that the promotion of EV-D68 replication by ADAR1p110 is also related to the inhibition of PKR activation by its dsRBDs. Our study illustrates that ADAR1p110 is a novel pro-viral factor of EV-D68 replication and provides a theoretical basis for EV-D68 antiviral research.

Respiratory and intestinal epithelial cells exhibit differential susceptibility and innate immune responses to contemporary EV-D68 isolates.

Enterovirus D68 (EV-D68) has been implicated in outbreaks of severe respiratory illness and is associated with acute flaccid myelitis (AFM). EV-D68 is often detected in patient respiratory samples but has also been detected in stool and wastewater, suggesting the potential for both respiratory and enteric routes of transmission. Here, we used a panel of EV-D68 isolates, including a historical pre-2014 isolate and multiple contemporary isolates from AFM outbreak years, to define the dynamics of viral replication and the host response to infection in primary human airway cells and stem cell-derived enteroids. We show that some recent EV-D68 isolates have decreased sensitivity to acid and temperature compared with earlier isolates and that the respiratory, but not intestinal, epithelium induces a robust type III interferon response that restricts infection. Our findings define the differential responses of the respiratory and intestinal epithelium to contemporary EV-D68 isolates and suggest that a subset of isolates have the potential to target both the human airway and gastrointestinal tracts.

Low circulation of Influenza A and coinfection with SARS-CoV-2 among other respiratory viruses during the COVID-19 pandemic in a region of southern Brazil.

With the arrival of coronavirus disease 2019 (COVID-19) in Brazil in February 2020, several preventive measures were taken by the population aiming to avoid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection including the use of masks, social distancing, and frequent hand washing then, these measures may have contributed to preventing infection also by other respiratory viruses. Our goal was to determine the frequencies of Influenza A and B viruses (FLUAV/FLUBV), human mastadenovirus C (HAdV-C), Enterovirus 68 (EV-68), and rhinovirus (RV) besides SARS-CoV-2 among hospitalized patients suspect of COVID-19 with cases of acute respiratory disease syndrome (ARDS) in the period of March to December 2020 and to detect possible coinfections among them. Nucleic acid detection was performed using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) in respiratory samples using naso-oropharyngeal swabs and bronchoalveolar lavage. A total of 418 samples of the 987 analyzed (42.3%) were positive for SARS-CoV-2, 16 (1.62%) samples were positive for FLUAV, no sample was positive for FLUBV or EV-68, 67 (6.78%) samples were positive for HAdV-C, 55 samples were positive for RV 1/2 (26.3%) and 37 for RV 2/2 (13.6%). Coinfections were also detected, including a triple coinfection with SARS-CoV-2, FLUAV, and HAdV-C. In the present work, a very low frequency of FLUV was reported among hospitalized patients with ARDS compared to the past years, probably due to preventive measures taken to avoid COVID-19 and the high influenza vaccination coverage in the region in which this study was performed.

Optimized photochemistry enables efficient analysis of dynamic RNA structuromes and interactomes in genetic and infectious diseases.

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (-) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases.

Enhanced Enterovirus D68 Replication in Neuroblastoma Cells Is Associated with a Cell Culture-Adaptive Amino Acid Substitution in VP1.

Since its emergence in the United States in 2014, enterovirus D68 (EV-D68) has been and is associated with severe respiratory diseases and acute flaccid myelitis. Even though EV-D68 has been shown to replicate in different neuronal cells in vitro, it is currently poorly understood which viral factors contribute to the ability to replicate efficiently in cells of the central nervous system and whether this feature is a clade-specific feature. Here, we determined the replication kinetics of clinical EV-D68 isolates from (sub)clades A, B1, B2, B3, and D1 in human neuroblastoma cells (SK-N-SH). Subsequently, we compared sequences to identify viral factors associated with increased viral replication. All clinical isolates replicated in SK-N-SH cells, although there was a large difference in efficiency. Efficient replication of clinical isolates was associated with an amino acid substitution at position 271 of VP1 (E271K), which was acquired during virus propagation in vitro Recognition of heparan sulfate in addition to sialic acids was associated with increased attachment, infection, and replication. Removal of heparan sulfate resulted in a decrease in attachment, internalization, and replication of viruses with E271K. Taken together, our study suggests that the replication kinetics of EV-D68 isolates in SK-N-SH cells is not a clade-specific feature. However, recognition of heparan sulfate as an additional receptor had a large effect on phenotypic characteristics in vitro. These observations emphasize the need to compare sequences from virus stocks with clinical isolates in order to retrieve phenotypic characteristics from original virus isolates.IMPORTANCE Enterovirus D68 (EV-D68) causes mild to severe respiratory disease and is associated with acute flaccid myelitis since 2014. Currently, the understanding of the ability of EV-D68 to replicate in the central nervous system (CNS), and whether it is associated with a specific clade of EV-D68 viruses or specific viral factors, is lacking. Comparing different EV-D68 clades did not reveal clade-specific phenotypic characteristics. However, we did show that viruses which acquired a cell culture-adapted amino acid substitution in VP1 (E271K) recognized heparan sulfate as an additional receptor. Recognition of heparan sulfate resulted in an increase in attachment, infection, and replication in neuroblastoma cells compared with viruses without this specific amino acid substitution. The ability of EV-D68 viruses to acquire cell culture-adaptive substitutions which have a large effect in experimental settings emphasizes the need to sequence virus stocks.

Mapping Attenuation Determinants in Enterovirus-D68.

Enterovirus (EV)-D68 has been associated with epidemics in the United Sates in 2014, 2016 and 2018. This study aims to identify potential viral virulence determinants. We found that neonatal type I interferon receptor knockout mice are susceptible to EV-D68 infection via intraperitoneal inoculation and were able to recapitulate the paralysis process observed in human disease. Among the EV-D68 strains tested, strain US/MO-14-18949 caused no observable disease in this mouse model, whereas the other strains caused paralysis and death. Sequence analysis revealed several conserved genetic changes among these virus strains: nucleotide positions 107 and 648 in the 5'-untranslated region (UTR); amino acid position 88 in VP3; 1, 148, 282 and 283 in VP1; 22 in 2A; 47 in 3A. A series of chimeric and point-mutated infectious clones were constructed to identify viral elements responsible for the distinct virulence. A single amino acid change from isoleucine to valine at position 88 in VP3 attenuated neurovirulence by reducing virus replication in the brain and spinal cord of infected mice.

Viral Etiology of Acute Lower Respiratory Tract Infections in Hospitalized Children in Nagasaki, a Regional City of Japan in 2013-2015.

BACKGROUND: Acute lower respiratory tract infections (ALRIs) are the most common disease for hospitalized children in Japan. The aim of this study was to identify viruses in children hospitalized for ALRIs and to demonstrate epidemiologic and clinical characteristics in Japan. METHODS: During a 2-year period from February 2013 to January 2015, we collected nasopharyngeal swab specimens from almost all hospitalized children with ALRIs in Nagasaki, a regional city of Japan, and its environs. Viruses were detected by multiplex polymerase chain reaction from these samples. RESULTS: We detected one or more viruses from 259 (69%) of 374 patients, 227 of whom were infected with a single virus, 30 with 2, and 2 with 3 viruses. Detected viruses in this study were rhinovirus (46.4%), respiratory syncytial virus (29.7%), human metapneumovirus (6.8%), parainfluenza virus (5.5%), enterovirus D68 (3.4%), influenza virus (2.7%), adenovirus (2.4%), bocavirus (2.0%) and Coxsackie virus (1.0%). We detected a seasonal shift in respiratory syncytial virus outbreaks from the 2013-2014 to the 2014-2015 seasons. There was no significant difference in clinical course and severity among those viruses. Patients with a history of asthma or underlying diseases were older and more frequently required oxygen therapy than previously healthy patients. CONCLUSIONS: Viral etiology in hospitalized children with ALRIs in Nagasaki, Japan, was similar to that in many other countries. Enterovirus D68, which was recently recognized as a causative agent of severe ALRIs, was also identified in this study area. Severity of ALRIs may depend on underlying disease rather than type of etiologic virus.

Typical Stress Granule Proteins Interact with the 3' Untranslated Region of Enterovirus D68 To Inhibit Viral Replication.

Stress granules (SGs) are formed in the cytoplasm under environmental stress, including viral infection. Human enterovirus D68 (EV-D68) is a highly pathogenic virus which can cause serious respiratory and neurological diseases. At present, there is no effective drug or vaccine against EV-D68 infection, and the relationship between EV-D68 infection and SGs is poorly understood. This study revealed the biological function of SGs in EV-D68 infection. Our results suggest that EV-D68 infection induced the accumulation of SG marker proteins Ras GTPase-activated protein-binding protein 1 (G3BP1), T cell intracellular antigen 1 (TIA1), and human antigen R (HUR) in the cytoplasm of infected host cells during early infection but inhibited their accumulation during the late stage. Simultaneously, we revealed that EV-D68 infection induces HUR, TIA1, and G3BP1 colocalization, which marks the formation of typical SGs dependent on protein kinase R (PKR) and eIF2α phosphorylation. In addition, we found that TIA1, HUR, and G3BP1 were capable of targeting the 3' untranslated regions (UTRs) of EV-D68 RNA to inhibit viral replication. However, the formation of SGs in response to arsenite (Ars) gradually decreased as the infection progressed, and G3BP1 was cleaved in the late stage as a strategy to antagonize SGs. Our findings have important implications in understanding the mechanism of interaction between EV-D68 and the host while providing a potential target for the development of antiviral drugs.IMPORTANCE EV-D68 is a serious threat to human health, and there are currently no effective treatments or vaccines. SGs play an important role in cellular innate immunity as a target with antiviral effects. This manuscript describes the formation of SGs induced by EV-D68 early infection but inhibited during the late stage of infection. Moreover, TIA1, HUR, and G3BP1 can chelate a specific site of the 3' UTR of EV-D68 to inhibit viral replication, and this interaction is sequence and complex dependent. However, this inhibition can be antagonized by overexpression of the minireplicon. These findings increase our understanding of EV-D68 infection and may help identify new antiviral targets that can inhibit viral replication and limit the pathogenesis of EV-D68.

Susceptibility of Enterovirus-D68 to RNAi-mediated antiviral knockdown.

Enterovirus D68 (EV-D68) represents an emerging pathogen which has demonstrated a capacity for causing epidemic illness in pediatric and immunocompromised patients. With no effective antiviral treatment available, therapeutic interventions are currently limited to supportive care. Utilizing available genomic sequences from the 2014 B3 Epidemic EV-D68 clade and the 1962 Fermon EV-D68 strains, we performed in silico comparative genomic analysis, identifying several islands of phylogenetic conservation within the viral RNA-dependent RNA polymerase gene. The effects of transfecting short-interfering double-stranded RNA (siRNA) molecules targeting these conserved sequences were tested in vitro using a human rhabdomyosarcoma cell-based model of EV-D68 infection. Two siRNA sequences demonstrated reproducible ability to abrogate EV-D68-mediated cytopathic effect in vitro. These siRNA sequences were also able to decrease EV-D68 genome replication, VP-2 capsid protein expression, and infectious particle production in vitro. EV-D68 knockdown was sequence-specific and not observed in cells treated with a negative control siRNA lacking sequence homology to the viral genome. The regions targeted by these siRNA's are located in highly conserved regions of the RNA-dependent RNA polymerase gene. The most potent siRNA targeted a sequence found in subsequent enzyme crystallographic studies to enhance the enzyme's thermostability (Wang et al., 2017). Topical nebulized siRNAs have recently been utilized as antivirals in human studies, with no adverse effects or toxicities noted (Gottlieb et al., 2016). Sequence selection is likely one primary factor determining the potential efficacy of such therapeutics. These results demonstrate that the identified siRNA sequences are able to suppress EV-D68 replication and cytopathic effect in vitro.

Bypassing pan-enterovirus host factor PLA2G16.

Enteroviruses are a major cause of human disease. Adipose-specific phospholipase A2 (PLA2G16) was recently identified as a pan-enterovirus host factor and potential drug target. In this study, we identify a possible mechanism of PLA2G16 evasion by employing a dual glycan receptor-binding enterovirus D68 (EV-D68) strain. We previously showed that this strain does not strictly require the canonical EV-D68 receptor sialic acid. Here, we employ a haploid screen to identify sulfated glycosaminoglycans (sGAGs) as its second glycan receptor. Remarkably, engagement of sGAGs enables this virus to bypass PLA2G16. Using cryo-EM analysis, we reveal that, in contrast to sialic acid, sGAGs stimulate genome release from virions via structural changes that enlarge the putative openings for genome egress. Together, we describe an enterovirus that can bypass PLA2G16 and identify additional virion destabilization as a potential mechanism to circumvent PLA2G16.

A case report of the enterovirus-D68 associated severe acute respiratory illness in a pediatric case from India.

Since, early 2000s, there have been several clusters of enterovirus-D68 (EV D68) associated respiratory illness reported from various countries. Recent largest and most wide-spread outbreak of EV-D68 associated severe acute respiratory illness (SARI) occurred in North America. Present report describes a case of EV-D68 associated severe acute respiratory illness from India with a whole genome sequence. The case was identified through retrospective analysis of Influenza SARI surveillance sample collected during September 2017 using Next Generation sequencing. EV D68 positive child aged two years and presented with asthma like symptoms for which he was admitted to ICU. The child tested negative for Influenza, RSV, Rhinovirus, PIV, hMPV and adenovirus, on real time RT-PCR. And on NGS full EV D68 genome was retrieved belonging to sub-clade B3. In ICU, child received anti-bacterial and anti-viral therapy. The child recovered with-out any sequelae and was discharged one week later. Present report highlights the importance of studying this emergent virus EV-D68 through prospective studies to understand the burden and epidemiological pattern in the country and its implications.

Enterovirus D68 Causing Acute Respiratory Infection: Clinical Characteristics and Differences With Acute Respiratory Infections Associated With Enterovirus Non-D68.

BACKGROUND: Enterovirus (EV) D68 is mainly associated with acute respiratory infection (ARI). Since 2014, when outbreaks in different countries were observed, this emerging virus was considered a potential threat to public health. METHODS: During 2015-2017, the presence of enterovirus RNA was investigated in all respiratory samples of children younger than 15 years of age with ARI, obtained for virologic studies in the Pediatric Emergency Care Units and wards of 2 hospitals in Gipuzkoa (Spain), using a commercial multiplex real-time polymerase chain reaction. When enterovirus was detected, a polymerase chain reaction to amplify a specific viral polyprotein (VP1) gene region of EV-D68 was performed. RESULTS: In 2016, EV-D68 circulation was associated to ARI, with the highest incidence in the spring months. EV-D68 was detected in 44 children, mean age 30.1 ± 31.7 months old, 23 (52.3%) of them females and 17 (38.6%) with underlying respiratory medical conditions. Thirty-two patients (72%) required hospital admission, receiving the discharge diagnosis of recurrent wheezing (37.5%), asthmatic crisis (37.5%) or bronchiolitis (12.5%). Seven children (15.9%) needed the support of the pediatric intensive care unit. When coinfections were excluded, children with EV-D68 infection presented with increased work of breathing, recurrent wheezing or asthmatic crisis, more frequently than those with ARI associated with EV non-D68. Moreover, clinical outcomes (hospitalization, respiratory support) were more severe. All 44 EV-D68 strains detected belonged to lineage B3. CONCLUSIONS: EV-D68 circulated widely in Gipuzkoa during 2016 and was associated with severe ARI. In children with severe ARI of unknown etiology, the presence of EV-D68 should be considered.

Contemporary Circulating Enterovirus D68 Strains Have Acquired the Capacity for Viral Entry and Replication in Human Neuronal Cells.

Enterovirus D68 (EV-D68) has historically been associated with respiratory illnesses. However, in the summers of 2014 and 2016, EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis/paralysis (AFM/AFP) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. To assess the potential neurotropism of EV-D68, we utilized the neuroblastoma-derived neuronal cell line SH-SY5Y as a cell culture model to determine if differential infection is observed for different EV-D68 strains. In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Viral replication and infectivity in SH-SY5Y were assessed using multiple assays: virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to viral binding and entry were at least partly responsible for the differential infectivity phenotype. Transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains that could not directly infect SH-SY5Y. In addition to supporting virus replication and other functional studies, this cell culture model may help identify the signatures of virulence to confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.IMPORTANCE Since the EV-D68 outbreak during the summer of 2014, evidence of a causal link to a type of limb paralysis (AFM) has been mounting. In this article, we describe a neuronal cell culture model (SH-SY5Y cells) in which a subset of contemporary 2014 outbreak strains of EV-D68 show infectivity in neuronal cells, or neurotropism. We confirmed the difference in neurotropism in vitro using primary human neuron cell cultures and in vivo with a mouse paralysis model. Using the SH-SY5Y cell model, we determined that a barrier to viral entry is at least partly responsible for neurotropism. SH-SY5Y cells may be useful in determining if specific EV-D68 genetic determinants are associated with neuropathogenesis, and replication in this cell line could be used as rapid screening tool for identification of neurotropic EV-D68 strains. This may assist with better understanding of pathogenesis and epidemiology and with the development of potential therapies.

Molecular diversity and biennial circulation of enterovirus D68: a systematic screening study in Lyon, France, 2010 to 2016.

BackgroundUnderstanding enterovirus D68 (EV-D68) circulation patterns as well as risk factors for severe respiratory and neurological illness is important for developing preventive strategies. Methods: Between 2010 and 2016, 11,132 respiratory specimens from hospitalised patients in Lyon, France, were screened for EV-D68 by PCR. Phylogenetic relationships of the viral-protein-1 sequences were reconstructed using maximum-likelihood and Bayesian-Markov-Chain-Monte-Carlo approaches. Results: Overall, 171 infections with a biennial pattern were detected, including seven, one, 55, none, 42, one and 65 cases annually during 2010-16. Children (< 16 years-old; n = 150) were mostly affected and 71% (n = 121) of the total patients were under 5 years-old. In 146 patients with medical reviews, 73% (n = 107) presented with acute respiratory distress. Among paediatric patients with medical reviews (n = 133), 55% (n=73) had an asthma/wheezing history, while among adults (n = 13), 11 had underlying diseases. In total, 45 patients had severe infections and 28 patients needed intensive care unit stays. No acute flaccid myelitis (AFM) was detected. We found genotypes A, B1, B2 B3 and D circulating, and no associations between these and clinical presentations. During the study, new genotypes continuously emerged, being replaced over time. We estimated that ancestors of currently circulating genotypes emerged in the late-1990s to 2010. Rises of the EV-D68 effective population size in Lyon coincided with infection upsurges. Phylogenetic analyses showed ongoing diversification of EV-D68 worldwide, coinciding with more infections in recent years and increases of reported AFM paediatric cases. Conclusions: Reinforcement of diagnostic capacities and clinical-based surveillance of EV-D68 infections is needed in Europe to assess the EV-D68 burden.

Surveillance for the identification of cases of acute respiratory infection by enterovirus D68 in children in a tertiary level care hospital during 2014-2016. / Vigilancia epidemiológica para la identificación de casos de infección respiratoria aguda por enterovirus D68 en niños en un hospital de tercer nivel de atención durante 2014-2016

Background: The reemergence of enterovirus D68 (EV-D68) infections in the United States was reported from August-October 2014 (691 cases). In Mexico, an outbreak at the National Institute of Respiratory Diseases was reported (24 cases). The results of epidemiological surveillance of Enterovirus sp. (EV) and other respiratory viruses in a national pediatric tertiary care level hospital are presented. Methods: Following the alert issued by the reemergence of EV-D68 in 2014, epidemiological surveillance -which only detected respiratory viruses by PCR in patients with influenza-like illness using nasopharyngeal swabs- expanded to include children with asthma exacerbation or acute respiratory distress. Positive samples to EV were confirmed and typed by sequencing. Subsequent sequencing was used to obtain the complete viral genome. Results: Of 1705 samples, 13 were positive to EV. Patients with EV presented the following comorbidities: chronic lung disease (7.7%), neoplastic disease (15.4%), allergic asthma/rhinitis (23%), recurrent pneumonia (23%), and other (23%). Of the 13 samples positive for EV, three were positive for EV-D68. These cases required invasive mechanical ventilation, presented no neurological involvement and survived. Conclusions: The impact of the population studied by EV-D68 was lower than that reported in Mexico during the same period. Cases of EV-D68 infection had multiple comorbidities, but few pulmonary comorbidities, which could explain the low attack rate. The epidemiological surveillance and infection prevention system may have contained the outbreak.

Introducción: La reemergencia de las infecciones por Enterovirus D68 (EV-D68) se reportó en los EE.UU. desde agosto-octubre de 2014 (691 casos). En México, un brote se reportó en el Instituto Nacional de Enfermedades Respiratorias (24 casos). Se presentan los resultados de la vigilancia epidemiológica en un hospital pediátrico nacional de tercer nivel para Enterovirus sp. (EV) y otros virus respiratorios. Método: Tras la alerta emitida por la reemergencia del EV-D68 en 2014, la vigilancia epidemiológica ­que solo detectaba virus respiratorios mediante PCR en pacientes con enfermedad tipo influenza mediante toma de hisopados nasofaríngeos­ se expandió para incluir niños con exacerbación de asma o dificultad respiratoria aguda. Las muestras positivas para EV fueron confirmadas y tipificadas por secuenciación. Posteriormente, se utilizó secuenciación de siguiente generación para obtener el genoma viral completo. Resultados: De 1705 muestras, 13 fueron positivas para EV. Los pacientes con EV presentaron la siguiente comorbilidad: enfermedad pulmonar crónica (7.7%), enfermedad neoplásica (15.4%), asma/rinitis alérgica (23%), neumonías de repetición (23%), y otras (23%). De las 13 muestras positivas para EV, tres resultaron positivas para EV-D68. Dichos casos requirieron ventilación mecánica invasiva, no tuvieron afectación neurológica y sobrevivieron. Conclusiones: La afectación por EV-D68 de la población estudiada fue menor que lo reportado en México durante el mismo periodo. Los casos de infección por EV-D68 presentan diversa comorbilidad, aunque escasas enfermedades pulmonares, lo cual pudiera explicar la baja tasa de ataque. La presencia del sistema de vigilancia epidemiológica establecido y la prevención de infecciones pudieron haber contenido el brote.

Vigilancia epidemiológica para la identificación de casos de infección respiratoria aguda por enterovirus D68 en niños en un hospital de tercer nivel de atención durante 2014-2016 / Surveillance for the identification of cases of acute respiratory infection by enterovirus D68 in children in a tertiary level care hospital during 2014-2016

Resumen Introducción: La reemergencia de las infecciones por Enterovirus D68 (EV-D68) se reportó en los EE.UU. desde agosto-octubre de 2014 (691 casos). En México, un brote se reportó en el Instituto Nacional de Enfermedades Respiratorias (24 casos). Se presentan los resultados de la vigilancia epidemiológica en un hospital pediátrico nacional de tercer nivel para Enterovirus sp. (EV) y otros virus respiratorios. Método: Tras la alerta emitida por la reemergencia del EV-D68 en 2014, la vigilancia epidemiológica -que solo detectaba virus respiratorios mediante PCR en pacientes con enfermedad tipo influenza mediante toma de hisopados nasofaríngeos- se expandió para incluir niños con exacerbación de asma o dificultad respiratoria aguda. Las muestras positivas para EV fueron confirmadas y tipificadas por secuenciación. Posteriormente, se utilizó secuenciación de siguiente generación para obtener el genoma viral completo. Resultados: De 1705 muestras, 13 fueron positivas para EV. Los pacientes con EV presentaron la siguiente comorbilidad: enfermedad pulmonar crónica (7.7%), enfermedad neoplásica (15.4%), asma/rinitis alérgica (23%), neumonías de repetición (23%), y otras (23%). De las 13 muestras positivas para EV, tres resultaron positivas para EV-D68. Dichos casos requirieron ventilación mecánica invasiva, no tuvieron afectación neurológica y sobrevivieron. Conclusiones: La afectación por EV-D68 de la población estudiada fue menor que lo reportado en México durante el mismo periodo. Los casos de infección por EV-D68 presentan diversa comorbilidad, aunque escasas enfermedades pulmonares, lo cual pudiera explicar la baja tasa de ataque. La presencia del sistema de vigilancia epidemiológica establecido y la prevención de infecciones pudieron haber contenido el brote.

Abstract Background: The reemergence of enterovirus D68 (EV-D68) infections in the United States was reported from August-October 2014 (691 cases). In Mexico, an outbreak at the National Institute of Respiratory Diseases was reported (24 cases). The results of epidemiological surveillance of Enterovirus sp. (EV) and other respiratory viruses in a national pediatric tertiary care level hospital are presented. Methods: Following the alert issued by the reemergence of EV-D68 in 2014, epidemiological surveillance -which only detected respiratory viruses by PCR in patients with influenza-like illness using nasopharyngeal swabs- expanded to include children with asthma exacerbation or acute respiratory distress. Positive samples to EV were confirmed and typed by sequencing. Subsequent sequencing was used to obtain the complete viral genome. Results: Of 1705 samples, 13 were positive to EV. Patients with EV presented the following comorbidities: chronic lung disease (7.7%), neoplastic disease (15.4%), allergic asthma/rhinitis (23%), recurrent pneumonia (23%), and other (23%). Of the 13 samples positive for EV, three were positive for EV-D68. These cases required invasive mechanical ventilation, presented no neurological involvement and survived. Conclusions: The impact of the population studied by EV-D68 was lower than that reported in Mexico during the same period. Cases of EV-D68 infection had multiple comorbidities, but few pulmonary comorbidities, which could explain the low attack rate. The epidemiological surveillance and infection prevention system may have contained the outbreak.