RNA Structure in the 5' Untranslated Region of Enterovirus D68 Strains with Differing Neurovirulence Phenotypes.

Enterovirus-D68 (EV-D68) is a positive-sense single-stranded RNA virus within the family Picornaviridae. EV-D68 was initially considered a respiratory virus that primarily affected children. However, in 2014, EV-D68 outbreaks occurred causing the expected increase in respiratory illness cases, but also an increase in acute flaccid myelitis cases (AFM). Sequencing of 2014 outbreak isolates revealed variations in the 5' UTR of the genome compared to the historical Fermon strain. The structure of the 5' UTR RNA contributes to enterovirus virulence, including neurovirulence in poliovirus, and could contribute to neurovirulence in contemporary EV-D68 strains. In this study, the secondary and tertiary structures of 5' UTR RNA from the Fermon strain and 2014 isolate KT347251.1 are analyzed and compared. Secondary structures were determined using SHAPE-MaP and TurboFold II and tertiary structures were predicted using 3dRNAv2.0. Comparison of RNA structures between the EV-D68 strains shows significant remodeling at the secondary and tertiary levels. Notable secondary structure changes occurred in domains II, IV and V. Shifts in the secondary structure changed the tertiary structure of the individual domains and the orientation of the domains. Our comparative structural models for EV-D68 5' UTR RNA highlight regions of the molecule that could be targeted for treatment of neurotropic enteroviruses.

Animal Models of Enterovirus D68 Infection and Disease.

Human enterovirus D68 (EV-D68) is a globally reemerging respiratory pathogen that is associated with the development of acute flaccid myelitis (AFM) in children. Currently, there are no approved vaccines or treatments for EV-D68 infection, and there is a paucity of data related to the virus and host-specific factors that predict disease severity and progression to the neurologic syndrome. EV-D68 infection of various animal models has served as an important platform for characterization and comparison of disease pathogenesis between historic and contemporary isolates. Still, there are significant gaps in our knowledge of EV-D68 pathogenesis that constrain the development and evaluation of targeted vaccines and antiviral therapies. Continued refinement and characterization of animal models that faithfully reproduce key elements of EV-D68 infection and disease is essential for ensuring public health preparedness for future EV-D68 outbreaks.

Transcriptomic Profiling Reveals a Role for TREM-1 Activation in Enterovirus D68 Infection-Induced Proinflammatory Responses.

Increasing cases related to the pathogenicity of Enterovirus D68 (EV-D68) have made it a growing worldwide public health concern, especially due to increased severe respiratory illness and acute flaccid myelitis (AFM) in children. There are currently no vaccines or medicines to prevent or treat EV-D68 infections. Herein, we performed genome-wide transcriptional profiling of EV-D68-infected human rhabdomyosarcoma (RD) cells to investigate host-pathogen interplay. RNA sequencing and subsequent experiments revealed that EV-D68 infection induced a profound transcriptional dysregulation of host genes, causing significantly elevated inflammatory responses and altered antiviral immune responses. In particular, triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in highly activated TREM-1 signaling processes, acting as an important mediator in EV-D68 infection, and it is related to upregulation of interleukin 8 (IL-8), IL-6, IL-12p70, IL-1ß, and tumor necrosis factor alpha (TNF-α). Further results demonstrated that NF-κB p65 was essential for EV-D68-induced TREM-1 upregulation. Moreover, inhibition of the TREM1 signaling pathway by the specific inhibitor LP17 dampened activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade, suggesting that TREM-1 mainly transmits activation signals to phosphorylate p38 MAPK. Interestingly, treatment with LP17 to inhibit TREM-1 inhibited viral replication and infection. These findings imply the pathogenic mechanisms of EV-D68 and provide critical insight into therapeutic intervention in enterovirus diseases.

Retrospective Study of the Upsurge of Enterovirus D68 Clade D1 among Adults (2014-2018).

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.

The pathogenesis and virulence of enterovirus-D68 infection.

In 2014, enterovirus D68 (EV-D68) emerged causing outbreaks of severe respiratory disease in children worldwide. In a subset of patients, EV-D68 infection was associated with the development of central nervous system (CNS) complications, including acute flaccid myelitis (AFM). Since then, the number of reported outbreaks has risen biennially, which emphasizes the need to unravel the systemic pathogenesis in humans. We present here a comprehensive review on the different stages of the pathogenesis of EV-D68 infection - infection in the respiratory tract, systemic dissemination and infection of the CNS - based on observations in humans as well as experimental in vitro and in vivo studies. This review highlights the knowledge gaps on the mechanisms of systemic dissemination, routes of entry into the CNS and mechanisms to induce AFM or other CNS complications, as well as the role of virus and host factors in the pathogenesis of EV-D68.

Enterovirus D68 molecular and cellular biology and pathogenesis.

In recent years, enterovirus D68 (EV-D68) has advanced from a rarely detected respiratory virus to a widespread pathogen responsible for increasing rates of severe respiratory illness and acute flaccid myelitis (AFM) in children worldwide. In this review, we discuss the accumulating data on the molecular features of EV-D68 and place these into the context of enterovirus biology in general. We highlight similarities and differences with other enteroviruses and genetic divergence from own historical prototype strains of EV-D68. These include changes in capsid antigens, host cell receptor usage, and viral RNA metabolism collectively leading to increased virulence. Furthermore, we discuss the impact of EV-D68 infection on the biology of its host cells, and how these changes are hypothesized to contribute to motor neuron toxicity in AFM. We highlight areas in need of further research, including the identification of its primary receptor and an understanding of the pathogenic cascade leading to motor neuron injury in AFM. Finally, we discuss the epidemiology of the EV-D68 and potential therapeutic approaches.

Three-Year Longitudinal Motor Function and Disability Level of Acute Flaccid Myelitis.

BACKGROUND: We summarize the long-term motor outcome and disability level in a cluster of pediatric patients with acute flaccid myelitis (AFM) associated with the enterovirus D68 outbreak in 2015. METHODS: This is a nationwide follow-up questionnaire analysis study. Clinical data including the motor function (manual muscle strength test) and other neurological symptoms were collected at the acute (nadir), recovery (six months), and chronic (three years) stages. We use the Barthel index, which measures 10 variables describing activity of daily living and mobility to assess the disability level. RESULTS: Clinical data of 33 patients with AFM (13 females, 20 males; median age = 4.1 years) were available. Among patients with tetraplegia or triplegia, paraplegia, and monoplegia at the acute stage, two of seven, four of thirteen, and two of thirteen exhibited complete recovery without paralysis; of those five of seven, eight of thirteen, and two of thirteen showed improvement with lesser limb involvement at the chronic stage, respectively. Nine patients (27%) demonstrated improvement at the recovery-to-chronic period. All six patients with positive isolation of enterovirus D68 from biological samples at the acute stage showed persistent motor deficits. Other neurological findings had better prognosis than motor weakness. Better Barthel index score at the chronic stage was observed (P < 0.001; median difference [95% confidence interval], 53 [40 to 63]), implying an improved disability level even in patients with persistent motor deficits. CONCLUSIONS: AFM has a high rate of persistent motor deficits showing one- to two-limb paralysis. Disability level of patients with AFM, however, generally improved at the three-year time point.

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.

Structure of the enterovirus D68 RNA-dependent RNA polymerase in complex with NADPH implicates an inhibitor binding site in the RNA template tunnel.

Enterovirus D68 (EV-D68) is an emerging viral pathogen belonging to the Enterovirus genus of the Picornaviridae family, which is a serious threat to human health and has resulted in significant economic losses. The EV-D68 genome encodes an RNA-dependent RNA polymerase (RdRp) 3Dpol, which is central for viral genome replication and considered as a promising target for specific antiviral therapeutics. In this study, we report the crystal structures of human EV-D68 RdRp in the apo state and in complex with the inhibitor NADPH, which was selected by using a structure-based virtual screening approach. The EV-D68-RdRp-NADPH complex is the first RdRp-inhibitor structure identified in the species Enterovirus D. The inhibitor NADPH occupies the RNA template binding channel of EV-D68 RdRp with a novel binding pocket. Additionally, residues involved in the NADPH binding pocket of EV-D68 RdRp are highly conserved in RdRps of enteroviruses. Therefore, the enzyme activity of three RdRps from EV-D68, poliovirus, and enterovirus A71 is shown to decrease when titrated with NADPH separately in vitro. Furthermore, we identified that NADPH plays a pivotal role as an RdRp inhibitor instead of a chain terminator during restriction of RNA-dependent RNA replication. In the future, derivatives of NADPH may pave the way for novel inhibitors of RdRp through compound modification, providing potential antiviral agents for treating enteroviral infection and related diseases.

Enterovirus D68 seroepidemiology in Taiwan, a cross sectional study from 2017.

BACKGROUND: Enterovirus D68 (EV-D68) was discovered in 1962 and has unique characteristics compared to the characteristics of other enteroviruses. There were few documented cases before the epidemic in the United States in 2014. The Taiwan Centers for Diseases Control also confirmed that EV-D68 has been endemic, and some cases of acute flaccid myelitis were reported in Taiwan. To understand the current EV-D68 serostatus, we performed an EV-D68 seroepidemiology study in Taiwan in 2017. METHODS: After informed consent was obtained, we enrolled preschool children, 6- to 15-year-old students and 16- to 49-year-old people. The participants underwent a questionnaire investigation and blood sampling to measure the EV-D68 neutralization antibody. RESULTS: In total, 920 subjects were enrolled from the northern, central, southern and eastern parts of Taiwan with a male-to-female ratio of 1.03. The EV-D68 seropositive rate was 32% (26/82) in infants, 18% (27/153) in 1-year-old children, 43% (36/83) in 2-year-old children, 60% (94/156) in 3- to 5-year-old children, 89% (108/122) in 6- to 11-year-old primary school students, 98% (118/121) in 12- to 15-year-old high school students, 100% (122/122) in 16- to 49-year-old women and 100% (81/81) in 16- to 49-year-old males in 2017. Among preschool children, EV-D68 seropositivity was related to age (p for trend <0.0001), size of household ≧4 members (p = 0.037) and kindergarten attendance (p = 0.027). The seropositive rate varied among different geographic regions. CONCLUSION: EV-D68 infection was prevalent, and its seropositive rates increased with age, larger household size and kindergarten attendance among preschool children.

Enterovirus D68 in a 6-year-old acute flaccid myelitis case in China, 2018: a case report.

BACKGROUND: Acute flaccid myelitis (AFM) are reported to be associated with enterovirus D68 infection. Though an increasing number of AFM cases were reported with EV-D68 infection in the US, few such cases have been found in China. CASE PRESENTATION: A 6-year-old boy presented with acute flaccid myelitis (AFM) involving left arm after fever and respiratory symptoms for 6 days. Computed Tomography (CT) revealed inflammation in both lungs and magnetic resonance imaging (MRI) of the brain and spine showed swelling in the left frontal lobe and brain stem. The patient was diagnosed with meningomyelitis. EV-D68 was detected from pharyngeal samples 36 days after the onset of the disease. CONCLUSION: We report the first EV-D68 infection in case of AFM in mainland China. AFM surveillance systems is recommended to be established in China to guide diagnosis, case reporting, and specimen collection and testing for better understanding its etiologies.

Mielitis flácida aguda por enterovirus-D68: A propósito de un caso / Acute flaccid myelitis due to enterovirus-D68. A case report

En 2014 el enterovirus D68 (EV-D68) surgió como un patógeno emergente en Estados Unidos y Canadá, responsable de la aparición de varios casos de mielitis flácida aguda en niños. Estos brotes se extendieron a nivel mundial, declarándose varios pacientes en España en 2015-2016, uno de ellos el que presentamos. Paciente de 22 meses con imposibilidad para elevar las extremidades superiores y para el sostén cefálico, reflejos osteotendinosos presentes. Analítica sanguínea, tóxicos en orina, tomografía axial computerizada (TAC) craneal y electroencefalograma normales, líquido cefalorraquídeo con discreta pleocitosis. En la resonancia magnética (RM) a las 24 horas se observa hiperseñal en T2 de C1-C7. El electromiograma evidencia patología periférica axonal motora. Se realiza nueva RM posteriormente que objetiva hipercaptación de raíces anteriores en la cauda equina. Los potenciales realizados muestran neuropatía óptica bilateral desmielinizante y afectación mixta de cordón posterior a nivel cervical. Se aísla EV-D68 en aspirado nasofaríngeo. La instauración de un déficit motor agudo es una urgencia médica en la que debemos considerar un amplio abanico de posibilidades. La mielitis flácida aguda (MFA) es un síndrome descrito en relación a los brotes producidos por EV-D68. Los hallazgos mencionados en nuestro paciente han sido descritos en relación a este diagnóstico

In 2014 enterovirus D68 (EV-D68) arose as an emerging pathogen in the United States and Canada, responsible for the appearance of several cases of acute flaccid myelitis in children. These flare-ups extended worldwide with several patients being declared in Spain in 2015-2016, one of whom we are presenting herein. A 22-month-old patient with impossibility to lift his upper limbs and head support, osteotendinous reflexes were present. Blood work, urine drugs, cranial computed tomography and electroencephalogram were normal, cerebral spinal fluid with discrete pleocytosis. In the magnetic resonance imaging (MRI) at 24 hours, T2 hypersignal of C1-C7 was observed. The electromyogram showed evidence of axonal motor peripheral pathology. A new MRI was performed subsequently which showed increased uptake by the anterior roots in cauda equina. The visual evoked potencial showed demyelinating bilateral optic neuropathy and mixed involvement of the posterior cord at the cervical level. EV-D68 was isolated from the nasopharyngeal aspirate. The onset of an acute motor deficit is a medical emergency in which we should consider a wide range of bilities. Acute flaccid myelitis is a syndrome described in relation with flare-ups produced by EV-D68. The findings mentioned in our patient have been described in relation with this diagnosis

Neurotropism of Enterovirus D68 Isolates Is Independent of Sialic Acid and Is Not a Recently Acquired Phenotype.

Acute flaccid myelitis (AFM) is a rare but serious illness of the nervous system, specifically affecting the gray matter of the spinal cord, motor-controlling regions of the brain, and cranial nerves. Most cases of AFM are pathogen associated, typically with poliovirus and enterovirus infections, and occur in children under the age of 6 years. Enterovirus D68 (EV-D68) was first isolated from children with pneumonia in 1962, but an association with AFM was not observed until the 2014 outbreak. Organotypic mouse brain slice cultures generated from postnatal day 1 to 10 mice and adult ifnar knockout mice were used to determine if neurotropism of EV-D68 is shared among virus isolates. All isolates replicated in organotypic mouse brain slice cultures, and three isolates replicated in primary murine astrocyte cultures. All four EV-D68 isolates examined caused paralysis and death in adult ifnar knockout mice. In contrast, no viral disease was observed after intracranial inoculation of wild-type mice. Six of the seven EV-D68 isolates, including two from 1962 and four from the 2014 outbreak, replicated in induced human neurons, and all of the isolates replicated in induced human astrocytes. Furthermore, a putative viral receptor, sialic acid, is not required for neurotropism of EV-D68, as viruses replicated within neurons and astrocytes independent of binding to sialic acid. These observations demonstrate that EV-D68 is neurotropic independent of its genetic lineage and can infect both neurons and astrocytes and that neurotropism is not a recently acquired characteristic as has been suggested. Furthermore, the results show that in mice the innate immune response is critical for restricting EV-D68 disease.IMPORTANCE Since 2014, numerous outbreaks of childhood infections with enterovirus D68 (EV-D68) have occurred worldwide. Most infections are associated with flu-like symptoms, but paralysis may develop in young children. It has been suggested that infection only with recent viral isolates can cause paralysis. To address the hypothesis that EV-D68 has recently acquired neurotropism, murine organotypic brain slice cultures, induced human motor neurons and astrocytes, and mice lacking the alpha/beta interferon receptor were infected with multiple virus isolates. All EV-D68 isolates, from 1962 to the present, can infect neural cells, astrocytes, and neurons. Furthermore, our results show that sialic acid binding does not play a role in EV-D68 neuropathogenesis. The study of EV-D68 infection in organotypic brain slice cultures, induced motor neurons, and astrocytes will allow for the elucidation of the mechanism by which the virus infection causes disease.

Enterovirus D68 Subclade B3 Circulation in Senegal, 2016: Detection from Influenza-like Illness and Acute Flaccid Paralysis Surveillance.

Following the 2014 outbreak, active surveillance of the EV-D68 has been implemented in many countries worldwide. Despite subsequent EV-D68 outbreaks (2014 and 2016) reported in many areas, EV-D68 circulation remains largely unexplored in Africa except in Senegal, where low levels of EV-D68 circulation were first noted during the 2014 outbreak. Here we investigate subsequent epidemiology of EV-D68 in Senegal from June to September 2016 by screening respiratory specimens from ILI and stool from AFP surveillance. EV-D68 was detected in 7.4% (44/596) of patients; 40 with ILI and 4 with AFP. EV-D68 detection was significantly more common in children under 5 years (56.8%, p = 0.016). All EV-D68 strains detected belonged to the newly defined subclade B3. This study provides the first evidence of EV-D68 B3 subclade circulation in Africa from patients with ILI and AFP during a 2016 outbreak in Senegal. Enhanced surveillance of EV-D68 is needed to better understand the epidemiology of EV-D68 in Africa.

Multi-season analyses of causative pathogens in children hospitalized with asthma exacerbation.

BACKGROUND: Respiratory viral and mycoplasma infections are associated with childhood asthma exacerbations. Here, we explored epidemiologic profile of causative pathogens and possible factors for exacerbation in a single center over a three-year period. METHODS: Hospitalized asthmatic children with attack aged 6 months-17 years were recruited between 2012 and 2015 (n = 216). Nasopharyngeal mucosa cell samples were collected from the participants and examined by reverse transcription-polymerase chain reaction to detect rhinovirus (RV), respiratory syncytial virus (RSV), enterovirus (EV), parainfluenza virus (PIV), Mycoplasma pneumoniae, and others. Clinical features, laboratory data, asthma exacerbation intensity, and asthma severity were compared among participants. Epidemiologic profile of causative pathogens and possible factors for exacerbation were explored. RESULTS: Viruses and/or Mycoplasma pneumoniae were detected in 75% of the participants. Rhinovirus (48%) was the most commonly detected virus in the participants with single infection, followed by RSV (6%). The median age at admission in the RV group was significantly higher than that in the RSV group. Insufficient asthma control and allergen sensitization were significantly related to RV-associated asthma exacerbation. There was no seasonality of pathogen types associated with asthma exacerbation although a sporadic prevalence of EV-D68 was observehinovirud. Rhinovirus were repeatedly detected in multiple admission cases. CONCLUSION: Our three-year analysis revealed that patients with RV infection were significantly prone to repeated RV infection in the subsequent exacerbation and good asthma control could prevent RV-associated asthma development and exacerbation. Multiple-year monitoring allowed us to comprehend the profile of virus- and/or mycoplasma-induced asthma exacerbation.

Current Understanding of Human Enterovirus D68.

Human enterovirus D68 (EV-D68), a member of the species Enterovirus D of the Picornaviridae family, was first isolated in 1962 in the United States. EV-D68 infection was only infrequently reported until an outbreak occurred in 2014 in the US; since then, it has continued to increase worldwide. EV-D68 infection leads to severe respiratory illness and has recently been reported to be linked to the development of the neurogenic disease known as acute flaccid myelitis (AFM), mostly in children, seriously endangering public health. Hitherto, treatment options for EV-D68 infections were limited to supportive care, and as yet there are no approved, specific antiviral drugs or vaccines. Research on EV-D68 has mainly focused on its epidemiology, and its virologic characteristics and pathogenesis still need to be further explored. Here, we provide an overview of current research on EV-D68, including the genotypes and genetic characteristics of recent epidemics, the mechanism of infection and virus-host interactions, and its relationship to acute flaccid myelitis (AFM), in order to broaden our understanding of the biological features of EV-D68 and provide a basis for the development of effective antiviral agents.

Serial MRI findings of acute flaccid myelitis during an outbreak of enterovirus D68 infection in Japan.

OBJECIVE: To clarify the neuroimaging findings of children with acute flaccid myelitis during an outbreak of EV-D68 infection. METHODS: We performed a detailed review of the spinal and cranial MRI results of 54 children with acute flaccid myelitis. We focused on the range of longitudinal lesions, the localization and appearance of lesions within a horizontal section, Gadolinium-enhancement, and changes over time. RESULTS: All children had longitudinal spinal lesions involving central gray matter. Twenty-six children had lesions spanning the entire spine. Six of them had weakness in all limbs, whereas seven had weakness of only one limb. Thirty-eight children had lesions in both gray and white matter and limb weakness tended to be more severe in these children. During the acute period, spinal lesions showed bilateral ill-defined widespread T2 hyperintensity. During the subacute period, lesions were well defined and confined to the anterior horn. The distribution of limb weakness was correlated with the appearance of lesions during the subacute period. Gadolinium enhancement was performed in 37 children, and enhancement was seen in the cauda equina in 29 children. Enhancement was infrequent within 2 days after onset but was seen in almost all children thereafter. Twenty-two children had brainstem lesions continuous with spinal lesions. CONCLUSION: Extensive longitudinal spinal lesions were characteristic in children with acute flaccid myelitis. Lesions were usually bilateral and widespread during the acute period, whereas localization to the anterior horn could become obvious. Although enhancement of the cauda equina was often observed, its appearance was sometimes delayed.