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Enterovirus D68 (EV-D68) is predominantly associated with mild respiratory infections, but can also cause severe respiratory disease and extra-respiratory complications, including acute flaccid myelitis. Systemic dissemination of EV-D68 is crucial for the development of extra-respiratory diseases, but it is currently unclear how EV-D68 spreads systemically (viremia). We hypothesize that immune cells contribute to the systemic dissemination of EV-D68, as this is a mechanism commonly used by other enteroviruses. Therefore, we investigated the susceptibility and permissiveness of human primary immune cells for different EV-D68 isolates. In human peripheral blood mononuclear cells inoculated with EV-D68, only B cells were susceptible but virus replication was limited. However, in B cell-rich cultures, such as Epstein-Barr virus-transformed B-lymphoblastoid cell line (BLCL) and primary lentivirus-transduced B cells, which better represent lymphoid B cells, were productively infected. Subsequently, we showed that dendritic cells (DCs), particularly immature DCs, are susceptible and permissive for EV-D68 infection and that they can spread EV-D68 to autologous BLCL. Altogether, our findings suggest that immune cells, especially B cells and DCs, could play an important role in the pathogenesis of EV-D68 infection. Infection of these cells may contribute to systemic dissemination of EV-D68, which is an essential step toward the development of extra-respiratory complications.IMPORTANCEEnterovirus D68 (EV-D68) is an emerging respiratory virus that has caused outbreaks worldwide since 2014. EV-D68 infects primarily respiratory epithelial cells resulting in mild respiratory diseases. However, EV-D68 infection is also associated with extra-respiratory complications, including polio-like paralysis. It is unclear how EV-D68 spreads systemically and infects other organs. We hypothesized that immune cells could play a role in the extra-respiratory spread of EV-D68. We showed that EV-D68 can infect and replicate in specific immune cells, that is, B cells and dendritic cells (DCs), and that virus could be transferred from DCs to B cells. Our data reveal a potential role of immune cells in the pathogenesis of EV-D68 infection. Intervention strategies that prevent EV-D68 infection of immune cells will therefore potentially prevent systemic spread of virus and thereby severe extra-respiratory complications., Competing Interests: The authors declare no conflict of interest.

Background: Enterovirus-D68 (EV-D68) predominantly causes respiratory disease. However, EV-D68 infections also have been associated with central nervous system (CNS) complications, most specifically acute flaccid myelitis (AFM). Diagnosing EV-D68-associated CNS disease is challenging since viral RNA is rarely detected in cerebrospinal fluid (CSF)., Objective: In order to determine an EV antibody index (AI), we evaluated the value of a commercially available quantitative ELISA to detect EV-specific antibodies in paired CSF and blood., Study Design: Nine paired CSF and blood samples were obtained from patients with EV-D68-associated AFM or from patients with a confirmed EV-associated CNS disease. EV-specific antibodies were detected using a quantitative ELISA. A Reiber diagram analysis was performed, by which the AI was calculated. Subsequently, EV ELISA results were compared with an EV-D68 virus neutralization test., Results: ELISA detected EV-specific antibodies in 1 out of the 3 patients with EV-D68-associated AFM and in 3 out of the 6 patients with confirmed EV-associated CNS disease. In these patients, the AI was indicative for intrathecal antibody production against enterovirus. Assay comparison showed that EV-D68 neutralizing antibody detection increased the sensitivity of EV-D68 antibody detection., Conclusions: A quantitative EV IgG ELISA in combination with Reiber diagram analysis and AI-calculation can be used as a diagnostic tool for EV-associated CNS disease, including EV-D68. An EV-D68 specific ELISA will improve the sensitivity of the tool. With the growing awareness that the detection of non-polio enteroviruses needs to be improved, diagnostic laboratories should consider implementation of EV serology., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2020 Sooksawasdi Na Ayudhya et al.)