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) 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.

Enterovirus D68 (EV-D68), as one of the major pathogens of paediatric respiratory disease, has been widely spread in the population in recent years. As the basis of virus antigenicity, antigenic epitopes are essential to monitoring the transformation of virus antigenicity. However, there is a lack of systematic studies on the antigenic epitopes of EV-D68. In this study, a bioinformatics-based prediction algorithm for human enteroviruses was used to predict the conformational epitopes of EV-D68. The prediction results showed that the conformational epitopes of EV-D68 were clustered into three sites: site 1, site 2, and site 3. Site 1 was located in the \"north rim\" region of the canyon near the fivefold axis; site 2 was located in the \"puff\" region near the twofold axis; and site 3 consisted of two parts, one in the \"knob\" region on the south rim of the canyon and the other in the threefold axis region. The predicted epitopes overlapped highly with the binding regions of four reported monoclonal antibodies (mAbs), indicating that the predictions were highly reliable. Phylogenetic analysis showed that amino acid mutations in the epitopes of the VP1 BC loop, DE loop, C-terminus, and VP2 EF loop played a crucial role in the evolutionary divergence of EV-D68 clades/subclades and epidemics. This finding indicated that the VP1 BC loop, DE loop, C-terminus, and VP2 EF loop were the most important epitopes of EV-D68. Research on the epitopes of EV-D68 will contribute to outbreak surveillance and to the development of diagnostic reagents and recombinant vaccines.

To investigate the seroepidemiological features of enterovirus D68 (EV-D68) in the healthy population from 2012 to 2017 in Beijing, China. A retrospective cross-sectional investigation was conducted using serum specimens collected from healthy individuals in Beijing from 2012 to 2017. These samples were tested for neutralization antibodies (NtAbs) against EV-D68. The sera from six EV-D68 infected patients in the acute or convalescent phase were used to determine the protection level of NtAbs against EV-D68. The geometric means of the titers (GMT) of EV-D68 NtAbs in 2012 and 2017 were 92.82 and 242.91, respectively; the seroprevalences of EV-D68 were 89.43% and 98.43%, respectively. The GMT reached its peak in the 11 to 15 age group in 2012, while in 16 to 20 age group in 2017. We also observed that EV-D68 NtAbs titers of six sera from the acute phase were all less than equal to  1:64 and that of three sera from the convalescent phase were all more than 1:64. Anti-EV-D68 NtAbs in the population remained low from 2012 to 2016 but increased significantly in 2017. Although most of the EV-D68 infections remain undetected in Beijing, the risk of a large outbreak of EV-D68 exists and should be taken seriously.

OBJECTIVE: As an immunofluorescence assay for enterovirus D68 (EV-D68) is not available in the enteroviruses surveillance network in Taiwan, EV-D68 may be the actual pathogen of untypeable enterovirus-suspected isolates.
METHODS: The untypeable isolates collected from 2007 through 2014 were identified by nucleic acid amplification-based methods and sequencing of the VP1 region to analyze the phylogeny and epidemiology of EV-D68 in Taiwan.
RESULTS: Twenty-nine EV-D68 isolates were sequenced, including 15 Cluster 3 and 14 Cluster 1 viruses. Approximately 41% of the patients were children under 5 years of age and their infections peaked in August. The ratio of male to female patients was 1.5 and 3.67 for Cluster 3 and Cluster 1, respectively. Fever and respiratory symptoms were commonly reported in EV-D68-infected patients. The results of phylogenetic analyses showed that EV-D68 isolates between 2007 and 2014 belonged to different clusters and existed for years, indicating that endemic circulation of EV-D68 existed in Taiwan.
CONCLUSIONS: This study showed that EV-D68 has been endemic in Taiwan for some years despite a small number of positive cases. The continuous monitoring and efforts towards the improvement of diagnostic techniques are required to complete the surveillance system. This study provided the genetic and epidemiological information which could contribute to understanding the etiology and epidemiology of EV-D68.