Enterovirus D68 (EV-D68) is an emerging pathogen that has caused outbreaks of severe respiratory disease worldwide, especially in children. We aim to investigate the prevalence and genetic characteristics of EV-D68 in children from Shanghai. Nasopharyngeal swab or bronchoalveolar lavage fluid samples collected from children hospitalized with community-acquired pneumonia were screened for EV-D68. Nine of 3997 samples were EV-D68-positive. Seven of nine positive samples were sequenced and submitted to GenBank. Based on partial polyprotein gene (3D) or complete sequence analysis, we found the seven strains belong to different clades and subclades, including three D1 (detected in 2013 and 2014), one D2 (2013), one D3 (2019), and two B3 (2014 and 2018). Overall, we show different clades and subclades of EV-D68 spread with low positive rates (0.2%) among children in Shanghai between 2013 and 2020. Amino acid mutations were found in the epitopes of the VP1 BC and DE loops and C-terminus; similarity analysis provided evidence for recombination as an important mechanism of genomic diversification. Both single nucleotide mutations and recombination play a role in evolution of EV-D68. Genetic instability within these clinical strains may indicate large outbreaks could occur following cumulative mutations.

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), a member of Enterovirus genus of the Picornaviridae family, mainly causes respiratory system-related diseases as well as neurological complications in some patients. At present, there is no effective vaccine or treatment for the virus. The aim of this research was to systematically analyse the molecular epidemiology, recombination and changes in the epitope of EV-D68 in China from 2008 to 2022. Through phylogenetic analysis based on VP1 sequences, it was found that there was limited information about EV-D68 infection before 2011 and that EV-D68 infection was dominated by the A2 gene subtype from 2011 to 2013 and the B3 genotype from 2014 to 2018, during which A2 and B3 were coprevalent and alternately prevalent. We also constructed a phylogenetic tree using the EV-D68 full-length genome sequences, and the genotype of each sequence was consistent with that of the VP1 sequence evolutionary tree. Recombination analysis showed that MH341715 underwent intertypic recombination with the A2 genotype MH341729 at the 5' untranslated region (5'UTR) and that P1-P3 underwent recombination with the B3 genotype MH341712. The capsid protein VP1 is one of the most important structural proteins. In VP1, the BC-loop (89-105 amino acids) and DE-loop (140-152 amino acids) are the most variable domains on the surface of the virus and are associated with epitopes. In this study, it was found that the dominant amino acid composition of the BC-loop and DE-loop continued to change with the epidemic of the virus; the amino acid composition also differed in different regions of the same genotypes. The ongoing genomic and molecular epidemiology of EV-D68 remains important for predicting emergence of new viruses and preventing major outbreaks of respiratory diseases.

Human enterovirus (EV) infections can lead to various manifestations, with variable correlations between genotypes and symptoms. Human enterovirus D68 (EV-D68) was considered to be associated with acute respiratory disease and acute flaccid myelitis. In this short report, both EV-D68 and poliovirus 3 were detected in the stool of a hospitalized 5-month child who presented with acute respiratory symptoms and who was recently vaccinated with oral polio vaccine (OPV), using a metatranscriptomic high-throughput sequencing method. The nearly full-length genome sequences with complete open reading frames of EV-D68 and poliovirus 3 were assembled. One previously-reported neurovirulence-related amino acid substitution (T860N) in the EV-D68 VP1 region was observed, but the patient showed no neurological symptoms. More attention should be paid to EV-D68, and continuous multiple syndrome-based surveillance on non-polio enterovirus is called for.

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.