Epidemiological and Clinical Insights into the Enterovirus D68 Upsurge in Europe 2021-2022 and Emergence of Novel B3-Derived Lineages, ENPEN Multicentre Study.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 infections and its clinical impact during the fall-winter season of 2021-2022. From 19 European countries, 58 institutes reported 10 481 (6.8%) EV-positive samples of which 1004 (9.6%) were identified as EV-D68 (including 852 respiratory samples). Clinical data were reported for 969 cases; 78.9% of infections were reported in children (0-5 years); and 37.9% of cases were hospitalized. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases including 6 diagnosed with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of 2 novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale European EV-D68 upsurge with severe clinical impact and the emergence of B3-derived lineages.

Enterovirus D68 infection upregulates SOCS3 expression to inhibit JAK-STAT3 signaling and antagonize the innate interferon response of the host.

Enterovirus D68 (EV-D68) can cause respiratory diseases and acute flaccid paralysis, posing a great threat to public health. Interferons are cytokines secreted by host cells that have broad-spectrum antiviral effects, inducing the expression of hundreds of interferon-stimulated genes (ISGs). EV-D68 activates ISG expression early in infection, but at a later stage, the virus suppresses ISG expression, a strategy evolved by EV-D68 to antagonize interferons. Here, we explore a host protein, suppressor of cytokine signaling 3 (SOCS3), is upregulated during EV-D68 infection and antagonizes the antiviral effects of type I interferon. We subsequently demonstrate that the structural protein of EV-D68 upregulated the expression of RFX7, a transcriptional regulator of SOCS3, leading to the upregulation of SOCS3 expression. Further exploration revealed that SOCS3 plays its role by inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). The expression of SOCS3 inhibited the expression of ISG, thereby inhibiting the antiviral effect of type I interferon and promoting EV-D68 transcription, protein production, and viral titer. Notably, a truncated SOCS3, generated by deleting the kinase inhibitory region (KIR) domain, failed to promote replication and translation of EV-D68. Based on the above studies, we designed a short peptide named SOCS3 inhibitor, which can specifically bind and inhibit the KIR structural domain of SOCS3, significantly reducing the RNA and protein levels of EV-D68. In summary, our results demonstrated a novel mechanism by which EV-D68 inhibits ISG transcription and antagonizes the antiviral responses of host type I interferon.

Contemporary enterovirus-D68 isolates infect human spinal cord organoids.

Enterovirus D68 (EV-D68) is a nonpolio enterovirus associated with severe respiratory illness and acute flaccid myelitis (AFM), a polio-like illness causing paralysis in children. AFM outbreaks have been associated with increased circulation and genetic diversity of EV-D68 since 2014, although the virus was discovered in the 1960s. The mechanisms by which EV-D68 targets the central nervous system are unknown. Since enteroviruses are human pathogens that do not routinely infect other animal species, establishment of a human model of the central nervous system is essential for understanding pathogenesis. Here, we describe two human spinal cord organoid (hSCO)-based models for EV-D68 infection derived from induced, pluripotent stem cell (iPSC) lines. One hSCO model consists primarily of spinal motor neurons, while the another model comprises multiple neuronal cell lineages, including motor neurons, interneurons, and glial cells. These hSCOs can be productively infected with contemporary strains, but not a historic strain, of EV-D68 and produce extracellular virus for at least 2 weeks without appreciable cytopathic effect. By comparison, infection with hSCO with another enterovirus, echovirus 11, causes significant structural destruction and apoptosis. Together, these findings suggest that EV-D68 infection is not the sole mediator of neuronal cell death in the spinal cord in those with AFM and that secondary injury from the immune response likely contributes to pathogenesis. IMPORTANCE AFM is a rare condition that causes significant morbidity in affected children, often contributing to life-long sequelae. It is unknown how EV-D68 causes paralysis in children, and effective therapeutic and preventative strategies are not available. Mice are not native hosts for EV-D68, and thus, existing mouse models use immunosuppressed or neonatal mice, mouse-adapted viruses, or intracranial inoculations. To complement existing models, we report two hSCO models for EV-D68 infection. These three-dimensional, multicellular models comprised human cells and include multiple neural lineages, including motor neurons, interneurons, and glial cells. These new hSCO models for EV-D68 infection will contribute to understanding how EV-D68 damages the human spinal cord, which could lead to new therapeutic and prophylactic strategies for this virus.

An automated high-throughput enterovirus D68 microneutralization assay platform.

Virus neutralization assays, widely used to detect and quantify antibodies induced by virus infection, are considered the gold standard for enterovirus serology testing. Conventional microneutralization assays have been used to assess enterovirus D68 (EV-D68) seroprevalence. While manual or automated 96-well assays are valuable, higher-density assays that increase throughput provide the opportunity to more efficiently screen large, population-based serology collections, as well as to test sample sets against multiple virus strains on the same plate or within the same run. Here, automation was implemented for bulk reagent dispensing, serial dilutions, and luminescence measurement to develop a 384-well enterovirus microneutralization assay that increases overall testing throughput, maintains the reproducibility of the standard 96-well assay, and reduces sample volume usage. EV-D68 strains Fermon, 14-18953, and 18-23087 were used to evaluate the automated 384-well microneutralization assay and compare to the conventional 96-well assay. Sensitivity and specificity were evaluated using pooled human sera and positive and negative control antisera. The Lower Limit of quantitation (LLOQ) was the same as for the 96-well assay and coefficients of variations (CV) of 7.35 %, 5.97 %, and 2.85 % for the three EV-D68 strains respectively, were well below the typical goal of ≤ 20 % CV for accuracy. Z-factor analysis yielded results of 0.694, 0.638, and 0.852, for the three EV-D68 strains respectively, indicating a high level of precision, reliability, and robustness. Intra-assay (7.25 %) and inter-assay (7.12 %) variability were well below 20 % CV. Moreover, the 96-well and 384-well versions of the assay were highly concordant, with a 0.955 correlation coefficient in titers obtained for 50 sera tested. Validation of this automated 384-well microneutralization will support its use in large serology screens assessing the presence of EV-D68 neutralizing antibodies in human populations.

Epidemiology and Sequence-Based Evolutionary Analysis of Circulating Non-Polio Enteroviruses.

Enteroviruses (EVs) are positive-sense RNA viruses, with over 50,000 nucleotide sequences publicly available. While most human infections are typically associated with mild respiratory symptoms, several different EV types have also been associated with severe human disease, especially acute flaccid paralysis (AFP), particularly with endemic members of the EV-B species and two pandemic types-EV-A71 and EV-D68-that appear to be responsible for recent widespread outbreaks. Here we review the recent literature on the prevalence, characteristics, and circulation dynamics of different enterovirus types and combine this with an analysis of the sequence coverage of different EV types in public databases (e.g., the Virus Pathogen Resource). This evaluation reveals temporal and geographic differences in EV circulation and sequence distribution, highlighting recent EV outbreaks and revealing gaps in sequence coverage. Phylogenetic analysis of the EV genus shows the relatedness of different EV types. Recombination analysis of the EV-A species provides evidence for recombination as a mechanism of genomic diversification. The absence of broadly protective vaccines and effective antivirals makes human enteroviruses important pathogens of public health concern.

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.