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.

Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 βB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.

Enterovirus D68 (EV-D68) is one of hundreds of non-polio enteroviruses that typically cause cold-like respiratory illness. The first EV-D68 outbreak in the United States in 2014 aroused widespread concern among the public and health authorities. The infection was found to be associated with increased surveillance of acute flaccid myelitis, a neurological condition that causes limb paralysis in conjunction with spinal cord inflammation. In vitro studies utilising two-dimensional (2D) and three-dimensional (3D) culture systems have been employed to elucidate the pathogenic mechanism of EV-D68. Various animal models have also been developed to investigate viral tropism and distribution, pathogenesis, and immune responses during EV-D68 infection. EV-D68 infections have primarily been investigated in respiratory, intestinal and neural cell lines/tissues, as well as in small-size immunocompetent rodent models that were limited to a young age. Some studies have implemented strategies to overcome the barriers by using immunodeficient mice or virus adaptation. Although the existing models may not fully recapitulate both respiratory and neurological disease observed in human EV-D68 infection, they have been valuable for studying pathogenesis and evaluating potential vaccine or therapeutic candidates. In this review, we summarise the methodologies and findings from each experimental model and discuss their applications and limitations.

Enterovirus D68 (EV-D68) is a re-emerging pathogen that is particularly common in children and may cause asthma-like respiratory infection and acute flaccid myelitis. However, in Korea, EV-D68 has never been reported thus far. This study aimed to identify EV-D68 from nasopharyngeal aspirates (NPAs) in Korean children with a respiratory tract infection.

The EV-D68 reference strain was purchased and blindly used to assess the detection ability of three commercial and one in-house mRT-PCR kit in 2018. Then, we selected children whose specimens were positive for human rhinovirus (HRV) and/or enterovirus (EV) by Allplex mRT-PCR (Seegene, Inc., Seoul, Korea) from April to December 2022. Total RNA was extracted from NPAs, and a partial 5'-UTR gene was amplified and sequenced for the identification of HRV/EV species. Additionally, PCR targeting the VP1 gene was performed to assess EV-D68-positive NPAs, followed by sequencing. Phylogenetic analysis and comparison of amino acid sequence alignments were performed using a partial VP1 gene of our and recent international EV-D68 strains.

Among the mRT-PCR kits tested, only the in-house kit was able to detect EV-D68 in 2018. However, we detected three EV-D68 strains among children hospitalized with fever and/or respiratory symptoms in September - December 2022 who tested positive for EV by the Allplex kit. Two of them were healthy toddlers with lower respiratory infections accompanied by new-onset wheezing but no neurologic complications. Among 34 children with lower respiratory infection who tested positive for HRV during the same period, EV-D68 was not detected. Phylogenetic analysis revealed that the first Korean EV-D68 belonged to subclade B3. Amino acid sequence alignment of international subclade B3 EV-D68 strains also showed that our strain is genetically more related to those from Europe than those from Japan.

We first detected EV-D68 in three Korean children who had EV detected by the Allplex mRT-PCR kit in 2022. EV-D68 also circulated in Korea in fall 2022, but the prevalence and severity seemed to be lower than those in previous reports from other countries.

Enterovirus-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.

EV-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.

Forty-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.

A 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.

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.

To review the epidemiology, clinical manifestations, and treatment strategies of nonpolio enterovirus and parechovirus (PeV) infections, and identify research gaps.

There is currently no approved antiviral agent for enterovirus or PeV infections, although pocapavir may be provided on a compassionate basis. Elucidation of the structure and functional features of enterovirus and PeV may lead to novel therapeutic strategies, including vaccine development.

Nonpolio human enterovirus and PeV are common childhood infections that are most severe among neonates and young infants. Although most infections are asymptomatic, severe disease resulting in substantial morbidity and mortality occurs worldwide and has been associated with local outbreaks. Long-term sequelae are not well understood but have been reported following neonatal infection of the central nervous system. The lack of antiviral treatment and effective vaccines highlight important knowledge gaps. Active surveillance ultimately may inform preventive strategies.

In patients with central nervous system (CNS) infections identification of the causative pathogen is important for treatment. Metagenomic next-generation sequencing techniques are increasingly being applied to identify causes of CNS infections, as they can detect any pathogen nucleic acid sequences present. Viromic techniques that enrich samples for virus particles prior to sequencing may simultaneously enrich ribosomes from bacterial pathogens, which are similar in size to small viruses.

We studied the performance of a viromic library preparation technique (VIDISCA) combined with low-depth IonTorrent sequencing (median ~ 25,000 reads per sample) for detection of ribosomal RNA from common pathogens, analyzing 89 cerebrospinal fluid samples from patients with culture proven bacterial meningitis.

Sensitivity and specificity to Streptococcus pneumoniae (n = 24) before and after optimizing threshold parameters were 79% and 52%, then 88% and 90%. Corresponding values for Neisseria meningitidis (n = 22) were 73% and 93%, then 67% and 100%, Listeria monocytogenes (n = 24) 21% and 100%, then 27% and 100%, and Haemophilus influenzae (n = 18) 56% and 100%, then 71% and 100%. A higher total sequencing depth, no antibiotic treatment prior to lumbar puncture, increased disease severity, and higher c-reactive protein levels were associated with pathogen detection.

We provide proof of principle that a viromic approach can be used to correctly identify bacterial ribosomal RNA in patients with bacterial meningitis. Further work should focus on increasing assay sensitivity, especially for problematic species (e.g. L. monocytogenes), as well as profiling additional pathogens. The technique is most suited to research settings and examination of idiopathic cases, rather than an acute clinical setting.

Enterovirus D68 (EV-D68) has recently been identified in biennial epidemics coinciding with diagnoses of non-polio acute flaccid paralysis/myelitis (AFP/AFM). We investigated the prevalence, genetic relatedness and associated clinical features of EV-D68 in 193 EV-positive samples from 193 patients in late 2018, UK. EV-D68 was detected in 83 (58 %) of 143 confirmed EV-positive samples. Sequencing and phylogenetic analysis revealed extensive genetic diversity, split between subclades B3 (n=50) and D1 (n=33), suggesting epidemiologically unrelated infections. B3 predominated in children and younger adults, and D1 in older adults and the elderly (P=0.0009). Clinical presentation indicated causation or exacerbation of respiratory distress in 91.4 % of EV-D68-positive individuals, principally cough (75.3 %), shortness of breath (56.8 %), coryza (48.1 %), wheeze (46.9 %), supplemental oxygen required (46.9 %) and fever (38.9 %). Two cases of AFM were observed, one with EV-D68 detectable in the cerebrospinal fluid, but otherwise neurological symptoms were rarely reported (n=4). Both AFM cases and all additional instances of intensive care unit (ICU) admission (n=5) were seen in patients infected with EV-D68 subclade B3. However, due to the infrequency of severe infection in our cohort, statistical significance could not be assessed.

A substantial amount of epidemiological data has been reported on Enterovirus D68 (EV-D68) infections after the 2014 outbreak. Our goal was to map the case fatality rate (CFR) and prevalence of current and past EV-D68 infections. We conducted a systematic review (PROSPERO, CRD42021229255) with published articles on EV-68 infections in PubMed, Embase, Web of Science and Global Index Medicus up to January 2021. We determined prevalences using a model random effect. Of the 4,329 articles retrieved from the databases, 89 studies that met the inclusion criteria were from 39 different countries with apparently healthy individuals and patients with acute respiratory infections, acute flaccid myelitis and asthma-related diseases. The CFR estimate revealed occasional deaths (7/1353) related to EV-D68 infections in patients with severe acute respiratory infections. Analyses showed that the combined prevalence of current and past EV-D68 infections was 4% (95% CI = 3.1-5.0) and 66.3% (95% CI = 40.0-88.2), respectively. The highest prevalences were in hospital outbreaks, developed countries, children under 5, after 2014, and in patients with acute flaccid myelitis and asthma-related diseases. The present study shows sporadic deaths linked to severe respiratory EV-D68 infections. The study also highlights a low prevalence of current EV-D68 infections as opposed to the existence of EV-D68 antibodies in almost all participants of the included studies. These findings therefore highlight the need to implement and/or strengthen continuous surveillance of EV-D68 infections in hospitals and in the community for the anticipation of the response to future epidemics.

Enterovirus D68 (EV-D68) is an emerging respiratory pathogen since the 2014 outbreak in the United States. A low level of virus circulation has been reported in Kuala Lumpur, Malaysia, in the past. However, the extent of the infection in Malaysia is not known. In the present study, we determine the seroepidemiology of EV-D68 in Kuala Lumpur, Malaysia, before and after the United States outbreak in August 2014. A luciferase-based seroneutralization test was developed using a clone-derived prototype Fermon strain carrying a nanoluciferase marker. We screened the neutralization capacity of 450 serum samples from children and adults (1-89 years old) collected between 2013 and 2015. EV-D68 seropositivity increased with age, with children aged 1-3 showing significantly lower seroprevalence compared to adults. Multivariate analysis showed that older age groups 13-49 years (odds ratio [OR] = 4.78; 95% confidence interval [CI] = 2.69-8.49; p < 0.0001) and ≥50 years (OR = 3.83; 95% CI = 2.19-6.68; p < 0.0001) were more likely to be EV-D68 seropositive than children <13 years. Sampling post-September 2014 compared to pre-Sept 2014 also predicted seropositivity (OR = 1.66; 95% CI = 1.04-2.65). The presence of neutralizing antibodies against EV-D68 in the study population suggests that EV-D68 was circulating before 2014. A higher seropositivity post-September 2014 suggests that Malaysia also experienced an upsurge in EV-D68 infections after the United States outbreaks in August 2014. A low seropositivity rate observed in children, especially those aged 1-3 years old, suggests that they are at risk and should be prioritized for future vaccination.

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.

Enterovirus D68 (EV-D68) is an emerging pathogen associated with respiratory diseases and/or acute flaccid myelitis. Here, two MAbs, 2H12 and 8F12, raised against EV-D68 virus-like particle (VLP), show distinct preference in binding VLP and virion and in neutralizing different EV-D68 strains. A combination of 2H12 and 8F12 exhibits balanced and potent neutralization effects and confers broader protection in mice than single MAbs when given at onset of symptoms. Cryo-EM structures of EV-D68 virion complexed with 2H12 or 8F12 show that both antibodies bind to the canyon region of the virion, creating steric hindrance for sialic acid receptor binding. Additionally, 2H12 binding can impair virion integrity and trigger premature viral uncoating. We also capture an uncoating intermediate induced by 2H12 binding, not previously described for picornaviruses. Our study elucidates the structural basis and neutralizing mechanisms of the 2H12 and 8F12 MAbs and supports further development of the 2H12/8F12 cocktail as a broad-spectrum therapeutic agent against EV-D68 infections in humans.

Acute flaccid myelitis (AFM) recently emerged in the United States as a rare but serious neurological condition since 2012. Enterovirus D68 (EV-D68) is thought to be a main causative agent, but limited surveillance of EV-D68 in the United States has hampered the ability to assess their causal relationship. Using surveillance data from the BioFire Syndromic Trends epidemiology network in the United States from January 2014 to September 2019, we characterized the epidemiological dynamics of EV-D68 and found latitudinal gradient in the mean timing of EV-D68 cases, which are likely climate driven. We also demonstrated a strong spatiotemporal association of EV-D68 with AFM. Mathematical modeling suggested that the recent dominant biennial cycles of EV-D68 dynamics may not be stable. Nonetheless, we predicted that a major EV-D68 outbreak, and hence an AFM outbreak, would have still been possible in 2020 under normal epidemiological conditions. Nonpharmaceutical intervention efforts due to the ongoing COVID-19 pandemic are likely to have reduced the sizes of EV-D68 and AFM outbreaks in 2020, illustrating the broader epidemiological impact of the pandemic.

The lack of active surveillance for enterovirus D68 (EV-D68) in the US has hampered the ability to assess the relationship with predominantly biennial epidemics of acute flaccid myelitis (AFM), a rare but serious neurological condition. Using novel surveillance data from the BioFire® Syndromic Trends (Trend) epidemiology network, we characterize the epidemiological dynamics of EV-D68 and demonstrate strong spatiotemporal association with AFM. Although the recent dominant biennial cycles of EV-D68 dynamics may not be stable, we show that a major EV-D68 epidemic, and hence an AFM outbreak, would still be possible in 2020 under normal epidemiological conditions. Significant social distancing due to the ongoing COVID-19 pandemic could reduce the size of an EV-D68 epidemic in 2020, illustrating the potential broader epidemiological impact of the pandemic.

Enterovirus D68 (EV-D68) infection has been associated with outbreaks of severe respiratory illness and increased cases of nonpolio acute flaccid myelitis. The patterns of EV-D68 circulation and molecular epidemiology are not fully understood. In this study, nasopharyngeal (NP) specimens collected from patients in the Lower Hudson Valley, New York, from 2014 to 2018 were examined for rhinovirus/enterovirus (RhV/EV) by the FilmArray respiratory panel. Selected RhV/EV-positive NP specimens were analyzed using two EV-D68-specific real-time RT-PCR assays, Sanger sequencing and metatranscriptomic next-generation sequencing. A total of 2,398 NP specimens were examined. EV-D68 was detected in 348 patients with NP specimens collected in 2014 (n = 94), 2015 (n = 0), 2016 (n = 160), 2017 (n = 5), and 2018 (n = 89), demonstrating a biennial upsurge of EV-D68 infection in the study area. Ninety-one complete or nearly complete EV-D68 genome sequences were obtained. Genomic analysis of these EV-D68 strains revealed dynamics and evolution of circulating EV-D68 strains since 2014. The dominant EV-D68 strains causing the 2014 outbreak belonged to subclade B1, with a few belonging to subclade B2. New EV-D68 subclade B3 strains emerged in 2016 and continued in circulation in 2018. Clade D strains that are rarely detected in the United States also arose and spread in 2018. The establishment of distinct viral strains and their variable circulation patterns provide essential information for future surveillance, diagnosis, vaccine development, and prediction of EV-D68-associated disease prevalence and potential outbreaks.