Enterovirus D68 Outbreak Research Articles

Inferring enterovirus D68 transmission dynamics from the genomic data of two 2022 North American outbreaks

Enterovirus D68 (EV-D68) has emerged as a significant cause of acute respiratory illness in children globally, notably following its extensive outbreak in North America in 2014. A recent outbreak of EV-D68 was observed in Ontario, Canada, from August to October 2022. Our phylogenetic analysis revealed a notable genetic similarity between the Ontario outbreak and a concurrent outbreak in Maryland, USA. Utilizing Bayesian phylodynamic modeling on whole genome sequences (WGS) from both outbreaks, we determined the median peak time-varying reproduction number (Rt) to be 2.70, 95% HPD (1.76, 4.08) in Ontario and 2.10, 95% HPD (1.41, 3.17) in Maryland. The Rt trends in Ontario closely matched those derived via EpiEstim using reported case numbers. Our study also provides new insights into the median infection duration of EV-D68, estimated at 7.94 days, 95% HPD (4.55, 12.8) in Ontario and 10.8 days, 95% HPD (5.85, 18.6) in Maryland, addressing the gap in the existing literature surrounding EV-D68’s infection period. We observed that the estimated Time since the Most Recent Common Ancestor (TMRCA) and the epidemic’s origin coincided with the easing of COVID-19 related social contact restrictions in both areas. This suggests that the relaxation of non-pharmaceutical interventions, initially implemented to control COVID-19, may have inadvertently facilitated the spread of EV-D68. These findings underscore the effectiveness of phylodynamic methods in public health, demonstrating their broad application from local to global scales and underscoring the critical role of pathogen genomic data in enhancing public health surveillance and outbreak characterization.

Real-Time Enterovirus D68 Outbreak Detection through Hospital Surveillance of Severe Acute Respiratory Infection, Senegal, 2023.

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

Retrospective Genotyping of Enteroviruses Using a Diagnostic Nanopore Sequencing Workflow.

Enteroviruses are among the most common viruses pathogenic to humans. They are associated with various forms of disease, ranging from mild respiratory illness to severe neurological diseases. In recent years, an increasing number of isolated cases of children developing meningitis or encephalitis as a result of enterovirus infection have been reported, as well as discrete enterovirus D68 outbreaks in North America in 2014 and 2016. We developed an assay to rapidly genotype enteroviruses by sequencing a region within the VP1 gene using nanopore Flongles. We retrospectively analyzed enterovirus-/rhinovirus-positive clinical samples from the Zurich, Switzerland area mainly collected during two seasons in 2019/2020 and 2021/2022. Respiratory, cerebrospinal fluid, and stool samples were analyzed. Whole-genome sequencing was performed on samples with ambiguous genotyping results and enterovirus D68-positive samples. Out of 255 isolates, a total of 95 different genotypes were found. A difference in the prevalence of enterovirus and rhinovirus infections was observed for both sample type and age group. In particular, children aged 0-4 years showed a higher frequency of enterovirus infections. Comparing the respiratory seasons, a higher prevalence was found, especially for enterovirus A and rhinovirus A after the SARS-CoV-2 pandemic. The enterovirus genotyping workflow provides a rapid diagnostic tool for individual analysis and continuous enterovirus surveillance.

Multimodal Surveillance Model for Enterovirus D68 Respiratory Disease and Acute Flaccid Myelitis among Children in Colorado, USA, 2022.

Surveillance for emerging pathogens is critical for developing early warning systems to guide preparedness efforts for future outbreaks of associated disease. To better define the epidemiology and burden of associated respiratory disease and acute flaccid myelitis (AFM), as well as to provide actionable data for public health interventions, we developed a multimodal surveillance program in Colorado, USA, for enterovirus D68 (EV-D68). Timely local, state, and national public health outreach was possible because prospective syndromic surveillance for AFM and asthma-like respiratory illness, prospective clinical laboratory surveillance for EV-D68 among children hospitalized with respiratory illness, and retrospective wastewater surveillance led to early detection of the 2022 outbreak of EV-D68 among Colorado children. The lessons learned from developing the individual layers of this multimodal surveillance program and how they complemented and informed the other layers of surveillance for EV-D68 and AFM could be applied to other emerging pathogens and their associated diseases.

Predicting the impact of COVID-19 non-pharmaceutical intervention on short- and medium-term dynamics of enterovirus D68 in the US

Recent outbreaks of enterovirus D68 (EV-D68) infections, and their causal linkage with acute flaccid myelitis (AFM), continue to pose a serious public health concern. During 2020 and 2021, the dynamics of EV-D68 and other pathogens have been significantly perturbed by non-pharmaceutical interventions against COVID-19; this perturbation presents a powerful natural experiment for exploring the dynamics of these endemic infections. In this study, we analyzed publicly available data on EV-D68 infections, originally collected through the New Vaccine Surveillance Network, to predict their short- and long-term dynamics following the COVID-19 interventions. Although long-term predictions are sensitive to our assumptions about underlying dynamics and changes in contact rates during the NPI periods, the likelihood of a large outbreak in 2023 appears to be low. Comprehensive surveillance data are needed to accurately characterize future dynamics of EV-D68. The limited incidence of AFM cases in 2022, despite large EV-D68 outbreaks, poses further questions for the timing of the next AFM outbreaks.

Clinical and molecular characteristics of the 2022 Enterovirus-D68 outbreak among hospitalized children, Ohio, USA

BackgroundEnterovirus-D68 (EV-D68) has appeared biennially in the United States following the 2014 outbreak. It has gained epidemiologic and clinical relevance and was identified as an important pathogen associated with severe respiratory and central nervous system diseases. We aim to describe the clinical and molecular characteristics of the post-pandemic 2022 Enterovirus-D68 outbreak in children evaluated in a tertiary pediatric hospital in Columbus, Ohio. MethodsEV-D68 RT-PCR was performed on nasopharyngeal specimens collected during Jun—Nov 2022 from children (<18 years), identified by 1) physician-order or 2) random selection of 10–15 specimens weekly that were Rhinovirus/Enterovirus-positive by physician-ordered respiratory virus panel. Patients who tested positive for EV-D68 were identified and clinical data and outcomes were analyzed. Partial viral VP1 region was sequenced and characterized. ResultsForty-four children positive for EV-D68 were identified, among which 88.6 % of patients presented with respiratory symptoms and 61.4 % required PICU admission. Two patients presented with AFM that was attributed to EV-D68. EV-D68 sequences from 2022 clustered within the B3 subclade. ConclusionsA significant proportion of children identified with EV-D68 during the 2022 outbreak had respiratory compromise requiring PICU admission. As the virus continues evolving, it is important to monitor the activity of EV-D68, characterizing these strains clinically and genetically, which will help to understand the viral pathogenicity and virulence.

Enterovirus D68 Outbreak in Children, Finland, August–September 2022

We observed an intense enterovirus D68 outbreak in children in southwest Finland in August-September 2022. We confirmed enterovirus D68 infection in 56 children hospitalized for respiratory illnesses and in 1 child with encephalitis but were not able to test all suspected patients. Continuing surveillance for enterovirus D68 is needed.

Qualitative detection of enterovirus D68 from PrimeStore® molecular transport medium: implications for home- and self-collection

To ensure proper specimen handling for detecting pathogens, like Enterovirus D68 (EV-D68), from home- and self-collection, alternative techniques are needed to ensure safe transport and reliable testing. PrimeStore® Molecular Transport Medium (MTM) may be an option since it does not require cold storage and inactivates virus while preserving RNA for detection. The purpose of this validation study was to demonstrate the ability to detect EV-D68 via rRT-PCR in MTM. Using a quantified EV-D68 positive control standard, MTM limit of detection for EV-D68 RNA is 104 cp/mL and RNA remains stable up to 30 days unfrozen. Positive and negative residual respiratory specimens from the 2018 EV-D68 outbreak were used for clinical testing. There was an 80% positive and 100% negative agreement with samples in MTM compared to reference. This study demonstrates the feasibility of EV-D68 detection from respiratory specimens collected and stored in PrimeStore® MTM, with implications for home- and self-collection.

Telaprevir Treatment Reduces Paralysis in a Mouse Model of Enterovirus D68 Acute Flaccid Myelitis.

In 2014, 2016, and 2018, the United States experienced unprecedented spikes in pediatric cases of acute flaccid myelitis (AFM), which is a poliomyelitis-like paralytic illness. Accumulating clinical, immunological, and epidemiological evidence has identified enterovirus D68 (EV-D68) as a major causative agent of these biennial AFM outbreaks. There are currently no available FDA-approved antivirals that are effective against EV-D68, and the treatment for EV-D68-associated AFM is primarily supportive. Telaprevir is an food and drug administration (FDA)-approved protease inhibitor that irreversibly binds the EV-D68 2A protease and inhibits EV-D68 replication in vitro. Here, we utilize a murine model of EV-D68 associated AFM to show that early telaprevir treatment improves paralysis outcomes in Swiss Webster (SW) mice. Telaprevir reduces both viral titer and apoptotic activity in both muscles and spinal cords at early disease time points, which results in improved AFM outcomes in infected mice. Following intramuscular inoculation in mice, EV-D68 infection results in a stereotypic pattern of weakness that is reflected by the loss of the innervating motor neuron population, in sequential order, of the ipsilateral (injected) hindlimb, the contralateral hindlimb, and then the forelimbs. Telaprevir treatment preserved motor neuron populations and reduced weakness in limbs beyond the injected hindlimb. The effects of telaprevir were not seen when the treatment was delayed, and toxicity limited doses beyond 35 mg/kg. These studies are a proof of principle, provide the first evidence of benefit of an FDA-approved antiviral drug with which to treat AFM, and emphasize both the need to develop better tolerated therapies that remain efficacious when administered after viral infections and the development of clinical symptoms. IMPORTANCE Recent outbreaks of EV-D68 in 2014, 2016, and 2018 have resulted in over 600 cases of a paralytic illness that is known as AFM. AFM is a predominantly pediatric disease with no FDA-approved treatment, and many patients show minimal recovery from limb weakness. Telaprevir is an FDA-approved antiviral that has been shown to inhibit EV-D68 in vitro. Here, we demonstrate that a telaprevir treatment that is given concurrently with an EV-D68 infection improves AFM outcomes in mice by reducing apoptosis and viral titers at early time points. Telaprevir also protected motor neurons and improved paralysis outcomes in limbs beyond the site of viral inoculation. This study improves understanding of EV-D68 pathogenesis in the mouse model of AFM. This study serves as a proof of principle for the first FDA-approved drug that has been shown to improve AFM outcomes and have in vivo efficacy against EV-D68 as well as underlines the importance of the continued development of EV-D68 antivirals.

Networking of public health and clinical laboratories: a successful strategy to uncover promptly two enterovirus D68 outbreaks in Lombardy region (northern Italy), July 2021-November 2022

Networking of public health and clinical laboratories: a successful strategy to uncover promptly two enterovirus D68 outbreaks in Lombardy region (northern Italy), July 2021-November 2022

An increase in enterovirus D68 circulation and viral evolution during a period of increased influenza like illness, The Johns Hopkins Health System, USA, 2022.

An increase in influenza like illness in children and adolescents at the Johns Hopkins Health system during summer 2022 was associated with increased positivity for enterovirus/ rhinovirus. We sought to characterize the epidemiology and viral evolution of enterovirus D68 (EV-D68). A cohort of remnant respiratory samples tested at the Johns Hopkins Microbiology Laboratory was screened for EV-D68. EV-D68 positives were characterized by whole genome sequencing and viral loads were assessed by droplet digital PCR (ddPCR). Genomic changes and viral loads were analyzed along with patients' clinical presentations. Of 566 screened samples, 126 were EV-D68 (22.3%). The median age of EV-D68 infected patients was four years, a total of 52 required supplemental oxygen (41.3%), and 35 (27.8%) were admitted. Lung disease was the most frequent comorbidity that was associated with hospitalization. A total of 75 complete and 32 partial genomes were characterized that made a new cluster within the B3 subclade that was closest to US genomes from 2018. Amino acid changes within the BC and DE loops were identified from 31 genomes (29%) which correlated with an increase in average viral load in respiratory specimens and the need for supplemental oxygen. EV-D68 outbreaks continue to cause influenza like illness that could be overwhelming for the health system due to a significant demand for high flow oxygen. Viral evolution and an increase in the susceptible population are likely driving the trends of the increased EV-D68 infections.

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.

Enterovirus D68 Infection in Human Primary Airway and Brain Organoids: No Additional Role for Heparan Sulfate Binding for Neurotropism.

ABSTRACTEnterovirus D68 (EV-D68) is an RNA virus that can cause outbreaks of acute flaccid paralysis (AFP), a polio-like disease. Before 2010, EV-D68 was a rare pathogen associated with mild respiratory symptoms, but the recent EV-D68 related increase in severe respiratory illness and outbreaks of AFP is not yet understood. An explanation for the rise in severe disease is that it may be due to changes in the viral genome resulting in neurotropism. In this regard, in addition to sialic acid, binding to heparan sulfate proteoglycans (HSPGs) has been identified as a feature for viral entry of some EV-D68 strains in cell lines. Studies in human primary organotypic cultures that recapitulate human physiology will address the relevance of these HSPG-binding mutations for EV-D68 infection in vivo. Therefore, in this work, we studied the replication and neurotropism of previously determined sialic acid-dependent and HSPG-dependent strains using primary human airway epithelial (HAE) cultures and induced human pluripotent stem cell (iPSC)-derived brain organoids. All three strains (B2/2042, B2/947, and A1/1348) used in this study infected HAE cultures and human brain organoids (shown for the first time). Receptor-blocking experiments in both cultures confirm that B2/2042 infection is solely dependent on sialic acid, while B2/947 and A1/1348 (HSPG to a lesser extent) binds to sialic acid and HSPG for cell entry. Our data suggest that HSPG-binding can be used by EV-D68 for entry in human physiological models but offers no advantage for EV-D68 infection of brain cells.IMPORTANCE Recent outbreaks of enterovirus D68, a nonpolio enterovirus, is associated with a serious neurological condition in young children, acute flaccid myelitis (AFM). As there is no antiviral treatment or vaccine available for EV-D68 it is important to better understand how EV-D68 causes AFM and why only recent outbreaks are associated with AFM. We investigated if a change in receptor usage of EV-D68 increases the virulence of EV-D68 in the airway or the central nervous system and thus could explain the increase in AFM cases. We studied this using physiologically relevant human airway epithelium and cerebral organoid cultures that are physiologically relevant human models. Our data suggest that heparan sulfate proteoglycans can be used by EV-D68 as an additional entry receptor in human physiological models but offers no advantage for EV-D68 infection of brain cells, and our data show the potential of these 46 innovative models for virology.

Circulation of enterovirus D68 (EV-D68) causing respiratory illness in New South Wales, Australia, between August 2018 and November 2019

The incidence of enterovirus D68 (EV-D68) in New South Wales, Australia, is unknown. As part of a state-wide surveillance program, enterovirus positive diagnostic specimens were assessed from patients presenting to hospitals with respiratory and meningitis syndromes from August 2018 to November 2019. Diagnostic enterovirus positive samples were collected from 339 patients and re-extracted followed by targeted PCR across the whole EV-D68 genome (7.4 kb). Obtained amplicons (n=208) were sequenced using Illumina sequencing technology and the phylogenetic relationships analysed relative to EV-D68 Fermon strain. We identified EV-D68 in 31 patients, both children (n=27) and adults (n=4). Phylogenetically, the majority (n=30) were from subclade B3, the same as that causing outbreaks of EV-D68 across the USA and Europe during 2018. These data strengthen the importance of having an active enterovirus surveillance network.

Evolution, geographic spreading, and demographic distribution of Enterovirus D68.

Worldwide outbreaks of enterovirus D68 (EV-D68) in 2014 and 2016 have caused serious respiratory and neurological disease. We collected samples from several European countries during the 2018 outbreak and determined 53 near full-length genome ('whole genome') sequences. These sequences were combined with 718 whole genome and 1,987 VP1-gene publicly available sequences. In 2018, circulating strains clustered into multiple subgroups in the B3 and A2 subclades, with different phylogenetic origins. Clusters in subclade B3 emerged from strains circulating primarily in the US and Europe in 2016, though some had deeper roots linking to Asian strains, while clusters in A2 traced back to strains detected in East Asia in 2015-2016. In 2018, all sequences from the USA formed a distinct subgroup, containing only three non-US samples. Alongside the varied origins of seasonal strains, we found that diversification of these variants begins up to 18 months prior to the first diagnostic detection during a EV-D68 season. EV-D68 displays strong signs of continuous antigenic evolution and all 2018 A2 strains had novel patterns in the putative neutralizing epitopes in the BC- and DE-loops. The pattern in the BC-loop of the USA B3 subgroup had not been detected on that continent before. Patients with EV-D68 in subclade A2 were significantly older than patients with a B3 subclade virus. In contrast to other subclades, the age distribution of A2 is distinctly bimodal and was found primarily among children and in the elderly. We hypothesize that EV-D68's rapid evolution of surface proteins, extensive diversity, and high rate of geographic mixing could be explained by substantial reinfection of adults. Better understanding of evolution and immunity across diverse viral pathogens, including EV-D68 and SARS-CoV-2, is critical to pandemic preparedness in the future.

Monitoring of Enterovirus D68 Outbreak in Israel by a Parallel Clinical and Wastewater Based Surveillance.

Enterovirus D68 (EVD68) was recently identified as an important cause of respiratory illness and acute flaccid myelitis (AFM), mostly in children. Here, we examined 472 pediatric patients diagnosed with severe respiratory illness and screened for EVD68 between April and October 2021. In parallel, samples collected from a wastewater treatment plant (WWTP) covering the residential area of the hospitalized patients were also tested for EVD68. Of the 472 clinical samples evaluated, 33 (7%) patients were positive for EVD68 RNA. All wastewater samples were positive for EVD68, with varying viral genome copy loads. Calculated EVD68 genome copies increased from the end of May until July 2021 and dramatically decreased at the beginning of August. A similar trend was observed in both clinical and wastewater samples during the period tested. Sequence analysis of EVD68-positive samples indicated that all samples originated from the same branch of subclade B3. This study is the first to use wastewater-based epidemiology (WBE) to monitor EVD68 dynamics by quantitative detection and shows a clear correlation with clinically diagnosed cases. These findings highlight the potential of WBE as an important tool for continuous surveillance of EVD68 and other enteroviruses.

Enterovirus D68-Associated Acute Respiratory Illness ─ New Vaccine Surveillance Network, United States, July-November 2018-2020.

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.

Enterovirus D68 in hospitalized children with respiratory symptoms in Guangdong from 2014 to 2018: Molecular epidemiology and clinical characteristics.

Enterovirus D68 (EV-D68) is an emerging pathogen in humans. EV-D68 causes a wide range of respiratory symptoms in children and has the propensity to cause severe complications. EV-D68 outbreaks are rarely investigated in mainland China. Therefore, in this study, we aimed to investigate the prevalence of EV-D68 in children and to describe the clinical manifestations as well as the phylogeny of EV-D68 in Guangdong Province from 2014 to 2018. Nasopharyngeal swabs were collected from hospitalized children with respiratory symptoms and screened for respiratory pathogens by fluorescence quantitative PCR and culture. The EV-positive samples were subsequently typed by sequencing the 5'-untranslated region and EV-D68-specific VP1 capsid gene. A phylogenetic tree was constructed by the maximum-likelihood method based on the VP1 gene using ClustalW. A total of 1,498 (59.8%) out of 2,503 children were screened positive for ≥1 virus species. Among the 158 (6.31%) EV-positive samples, 17 (0.68%) were identified as EV-D68. Most EV-D68 cases (n=14) were diagnosed with pneumonia and bronchial pneumonia. No deaths were found in EV-D68 cases. Wheezing occurred in EV-D68 cases more frequently (70.59% vs. 43.26%, P=0.040) than that of other EVs. All the EV-D68 were of clade B3, which were highly similar to the strains circulating in China. EV-D68 was the predominant enterovirus type in hospitalized children with respiratory symptoms in Guangdong Province. All the EV-D68 strains belong to clade B3. The development of diagnostic tools is warranted in order to monitor EV-D68 infections in China.

#16: Enterovirus D68 Visualized in the Anterior Horn of the Spinal Cord of a Pediatric Patient with Flaccid Paralysis

Abstract Background Acute flaccid myelitis (AFM) is a polio-like paralyzing illness of children. AFM incidence is increasing during every other year outbreaks that occur in the United States simultaneously with outbreaks of enterovirus D68 (EV-D68) infection. Demonstrating that EV-D68 directly causes AFM has been challenging due to rare detection of the virus in the cerebrospinal fluid (CSF) of patients despite frequent detection at nonsterile sites. Murine studies have shown that EV-D68 can infect spinal cord anterior horn motor neurons and cause paralysis, similar to poliovirus. However, a key outstanding question is whether EV-D68 causes AFM in humans by direct viral pathogenesis or by indirect host immunopathogenesis. Methods We investigated the pathogenesis of AFM using tissues from a previously reported case of a 5-year-old boy who presented in fall 2008 with four days of progressive limb and voice weakness followed by incontinence, apnea, and death. He had a CSF pleocytosis of 2094/µL with EV-D68 identified in the CSF by sequencing of the VP1 gene. We designed probes for in situ hybridization (ISH) based on this sequence to stain formalin fixed paraffin embedded tissues from his autopsy. For immunohistochemistry (IHC) we used both commercial polyclonal anti-EV-D68 antibodies and our own human monoclonal antibodies that stain virus infected cells in vitro. Immunophenotyping was done by IHC. To analyze gene transcription in the inflammatory transcriptome of these infected areas of spinal cord we used the GeoMx platform from Nanostring. Results We identified EV-D68 in the anterior horn of the patient’s spinal cord, corresponding to the location of motor neuron cell bodies. This area was highly inflamed, with an infiltrate of CD8 T cells and many macrophages. Viral RNA (see figure) and viral protein was visualized in motor neurons but not supporting cells using ISH and IHC, respectively. Viral RNA but not viral protein was detected rarely in the lungs in macrophages, which had extensive inflammatory infiltrate. The infiltrate was predominantly composed of macrophages with a CD8 T cell component as well. The transcriptome of cells in the inflamed tissue was enriched for genes involved in antigen presentation on MHC. Conclusions Deaths in AFM patients are rare and often distant from initial presentation, but this patient died four days after onset of weakness, allowing us to directly demonstrate that EV-D68 can infect the human spinal cord. Motor neurons but not neural support cells are directly infected by EV-D68 with a corresponding infiltrate of macrophages and CD8 T cells. Antigen presentation processes are upregulated in inflamed tissues. Therefore, both direct viral pathology and immune factors likely contribute to AFM disease in EV-D68 infection.

Enteroviruses in Respiratory Samples from Paediatric Patients of a Tertiary Care Hospital in Germany.

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.