EV-D68 is mainly divided into four subtypes and many branches, including the original classical subtypes and recent epidemic-related branches: Clade A, Clade B, Clade C, and Clade D (Figure 2). The location and corresponding year of specimen collection of EV-D68 VP1 sequences have revealed that EV-D68 subtype Clade A, Clade B, and Clade C are prevalent in multiple regions of American, Europe, and Asia. Several viral subtypes have often co-circulated, but Clade C is the least common of the subtypes. The major 2014 outbreak and currently prevalent strains in the United States are represented by Clade A1, Clade B1, Clade B4, and Clade B5 subtypes, whereas, also in 2014, European countries, such as Italy and the Netherlands, found confirmed cases of Clade A1, Clade A2(which has been reclassified to Clade D since 2016), Clade B1, and Clade B2 infection. In Canada, the major prevalent strain has been Clade B2. In Asian countries such as Thailand and Japan, the detection of Clade B and Clade C have been reported, whereas the main subtypes in China have been Clade A2 (which has been reclassified to Clade D since 2016) and Clade B2 [16]. The emergence of EV-D68 subclade D1 was recently reported in France and northern Italy, from August to November, 2018 [17,18]. Xiang et al. [18] reported that genogroup replacement has occurred in China since 2006. Before August 2011, Clade A was predominant in EV-D68-infected cases in China, and other strains were only scattered. Clade B emerged in October 2011 and co-circulated with Clade A until November 2013. However, in 2014, the circulating strains shifted from Clade A to B [19]. Genogroup replacement is one of the major causes of new outbreaks; fortunately, the genogroup replacement in China did not incur an EV-D68 upswing [19].

 

Apart from the virus itself, community immunity, and especially the pre-existing virus serum neutralizing antibody (NAbs) in the community, are very likely to influence the spread and prevalence of the virus. Research on viral NAbs of specific populations can trace the history of viral infection and predict the susceptibility of a population to certain viruses. Xiang et al. [20] collected serum samples from individuals with EV-D68 infection in Beijing, China from 2004 to 2011, tested the seroprevalence of EV-D68 NAbs, and found that the level of EV-D68 NAbs in the Chinese community in 2004 was remarkably lower in all age groups than in 2009. Furthermore, from 2007 to 2011, the NAbs against EV-D68 in the population increased over time. Meanwhile, researchers also found that the NAbs actually can inhibit virus replication. Thus, the high level of EV-D68 NAbs in the Chinese community could be one of the reasons why EV-D68 infection in China appears only sporadically instead of in the form of outbreaks.

Recombination and mutations caused by the error-prone polymerase, human immune response, and population polymorphism have been recognized as the main mechanisms for enterovirus evolution [21]. It has been reported that the US EV-D68 outbreak in 2014 was caused by a novel clade [7,22]; researchers detected six kinds of gene mutation, M291T, V341A, T860N, D297N, S1108G, and R2005K in a distinct clade B1 of EV-D68 strains, which were associated with the neurovirulence of acute flaccid myelitis (AFM) in American strains [7,22]. Moreover, through comparative analyses of EV-D68 2014 outbreak isolate sequences, Zhang et al. also identified that AFM/AFP-associated isolates belong to a single phylogenetic subclade, B1, with 12 substitutions that carry the same residues observed at equivalent positions in paralysis-causing enterovirus such as poliovirus, EV-D71, and EV-A71 [23]. Moreover, subclade B3, now the most prevalent EV-D68, is also reported to be associated with AFM [24]. Later, Huang et al. discovered three variants (C187T, C3277A, and A4020G) that differ from those previously identified through comparative genomic analysis. C3277A and A4020G cause amino acid substitutions T860N and S1108G, respectively, which are cleavage sites for the viral proteases 2A and 3C. The mutation of these sites alters the cleavage efficiency, leading to an increased rate of viral transmission and replication [5]. However, there have been cases of infection by EV-D68 strains with neurovirulence-related mutations that have only shown non-neurologic symptoms such as respiratory illness [7]. This result suggests that viral gene polymorphism is necessary to cause nervous system symptoms such as AFM, but other factors also need to be considered. Nevertheless, only one case was reported in China in 2011 in which the individual was infected by the mutant strain that had the six substitutions mentioned above. In 2014, a unique R220A mutation was detected in the EV-D68 strain prevalent in China [18], which might also result in changes in virulence. As in other enteroviruses, the loop sequences of the X and Y domains in the 3’-UTR of EV-D68 are complementary, but Xiang et al. found that the X loop sequences of the US 2014 outbreak EV-D68 strains were not complementary but instead identical to loop sequences of the Y domain, indicating that the structure of the 3’-UTR of the viral genome can affect viral replication. Meanwhile, all the epidemic EV-D68 American strains have C-U, C-A mutations in the 3’-UTR, a finding that may influence the phenotypes of the virus [18].

Existing epidemiological evidence indicates that the US has the highest prevalence of EV-D68. However, in recent years, cases of EV-D68 infection in Europe, Asia, and other countries have continued to increase. The evolution of EV-D68 exhibits geographic and temporal diversity, and changes in genotype largely affect the pathogenicity and epidemic characteristics of the virus.

Conclusions