Enterovirus-D68 - A Reemerging Non-Polio Enterovirus that Causes Severe Respiratory and Neurological Disease in Children

Abstract

The past decade has seen the global reemergence and rapid spread of enterovirus D68 (EV-D68), a respiratory pathogen that causes severe respiratory illness and paralysis in children. EV-D68 was first isolated in 1962 from children with pneumonia. Sporadic cases and small outbreaks have been reported since then with a major respiratory disease outbreak in 2014 associated with an increased number of children diagnosed with polio-like paralysis. From 2014-2018, major outbreaks have been reported every other year in a biennial pattern with > 90% of the cases occurring in children under the age of 16. With the outbreak of SARS-CoV-2 and the subsequent COVID-19 pandemic, there was a significant decrease in the prevalence EV-D68 cases along with other respiratory diseases. However, since the relaxation of pandemic social distancing protocols and masking mandates the number of EV-D68 cases have begun to rise again - culminating in another outbreak in 2022. Here we review the virology, pathogenesis, and the immune response to EV-D68, and discuss the epidemiology of EV-D68 infections and the divergence of contemporary strains from historical strains. Finally, we highlight some of the key challenges in the field that remain to be addressed.

Keywords: EV-D68; Enterovirus; acute Flaccid Myelitis; respiratory distress.

Figure 1.. Enterovirus-D68 Genome and capsid.

a and b) EV-D68 has an icosahedral, non-enveloped capsid consisting of 4 structural proteins, VP1, VP2, and VP3 on the external side of the capsid and VP4 on the internal side. This capsid surrounds the +ssRNA naked genome attached to VPg. c) The EV-D68 genome encodes for 4 structural proteins (VP1 – 4) and 7 non-structural proteins (2A - C and 3A - D). The internal ribosome entry site (IRES) is at the 5’ end and a poly-A tail terminates the 3’ end. The genome is ~7.2 kb in size and is composed of a single open reading frame (ORF). Initially, the polyprotein is processed into 3 precursor proteins, P1–P3. P1 is later proteolytically cleaved into the 4 structural proteins (VP1 – 4) while P2 and P3 are processed into replicase, VPg, proteases (2A and 3C), a polymerase (3D), and other non-structural proteins.

Figure 2.. Enterovirus-D68 Life Cycle.

EV-D68 attaches to the host cell membrane and is internalized through receptor-mediated endocytosis. The viral capsid undergoes uncoating and creates a pore through the endosomal membrane and the +ssRNA viral genome is released into the cytoplasm. The +ssRNA is translated into a polyprotein that is cleaved by host and viral proteases to generate structural (VP1 – 4) and non-structural (2A - C and 3A - D) proteins. During viral replication, −ssRNA is created and used as a template for new viral genome copies, which occurs on vesicular structures known as replication organelles. Progeny virions are assembled with structural proteins and VPg-linked RNA. The immature viral particles are then mostly taken up by autophagosomes where the acidic environment triggers viral capsid maturation. Virus is then released by either exocytosis of the autophagic vesicles (53) or by cell lysis. The figure was made using BioRender.com under license to CG.

Figure 3.. Number of EV-D68 reported by year.

Data reported by the National Enterovirus Surveillance System was used.

Figure 4.. Phylogenetic Tree based on Picornavirus VP1 sequence.

The NCBI Virus database was used to download all available Picornavirus VP1 capsid protein reference sequences (68). The sequences were then aligned using the online Clustal Omega Multiple Sequence Alignment Tool (69). The output from Clustal Omega was then transferred to Simple Phylogeny to create the phylogenetic tree (70). The results were then uploaded to iTOL for annotation (71). The genus enterovirus (shown in red) is genetically similar and descended from a common ancestor based on their VP1 capsid sequence.

Figure 5.. Enterovirus Phylogenetic Tree based on VP1 sequence.

The NCBI Virus database was used to download all available enterovirus VP1 capsid protein reference sequences (68). The sequences were then aligned using the Clustal Omega Multiple Sequence Alignment Tool (69). The resulting output from Clustal Omega was then transferred to the online tool, ATGCPhyl, to create the phylogenetic tree with bootstrap values. One thousand Bootstrap replicates were computed to estimate the accuracy of the phylogenetic tree (72). The results were then annotated in PowerPoint. EV-D68 (shown in red) is more genetically similar to rhinoviruses (Rhinovirus B and C) than other enterovirus species (Enterovirus C and D).

Figure 6.. NextStrain based EV-D68 Phylogenetic Tree.

A time-scaled phylogenetic tree was visualized using NextStrain based on the VP1 capsid protein sequence from 1992 to 2022 (76). The year that the EV-D68 isolate was detected is represented on the x-axis. The clade for each isolate is denoted by color per the legend (Clade A in blue, Clade B in green, yellow, and orange, and Clade C in red. Clade D is not represented). Major clade branches are labeled at the branch points. EV-D68 clade B1 was the major clade for strains identified in the 2014 outbreak, but a shift has been seen with clades A2 and B3 currently circulating.

Figure 7.. Enterovirus VP1 Protein Alignment

The NCBI Virus database was used to download all available enterovirus VP1 capsid protein reference sequences (68). The sequences were then aligned using the Clustal Omega Multiple Sequence Alignment Tool (69). The resulting output was then transferred to the online tool, NCBI Multiple Alignment. EV-D68 was selected as the anchor sequence, the top row, and BLOSUM62 was used for analysis and coloring (68). Amino acids identical to the anchor sequence (EV-D68) are represented by a dot and shown in blue, while amino acids not identical to the anchor sequence are shown in green a, with blue/green coloring indicating that the substitution is conservative.

Figure 7.. Enterovirus VP1 Protein Alignment

The NCBI Virus database was used to download all available enterovirus VP1 capsid protein reference sequences (68). The sequences were then aligned using the Clustal Omega Multiple Sequence Alignment Tool (69). The resulting output was then transferred to the online tool, NCBI Multiple Alignment. EV-D68 was selected as the anchor sequence, the top row, and BLOSUM62 was used for analysis and coloring (68). Amino acids identical to the anchor sequence (EV-D68) are represented by a dot and shown in blue, while amino acids not identical to the anchor sequence are shown in green a, with blue/green coloring indicating that the substitution is conservative.

Figure 7.. Enterovirus VP1 Protein Alignment

The NCBI Virus database was used to download all available enterovirus VP1 capsid protein reference sequences (68). The sequences were then aligned using the Clustal Omega Multiple Sequence Alignment Tool (69). The resulting output was then transferred to the online tool, NCBI Multiple Alignment. EV-D68 was selected as the anchor sequence, the top row, and BLOSUM62 was used for analysis and coloring (68). Amino acids identical to the anchor sequence (EV-D68) are represented by a dot and shown in blue, while amino acids not identical to the anchor sequence are shown in green a, with blue/green coloring indicating that the substitution is conservative.

Figure 7.. Enterovirus VP1 Protein Alignment

The NCBI Virus database was used to download all available enterovirus VP1 capsid protein reference sequences (68). The sequences were then aligned using the Clustal Omega Multiple Sequence Alignment Tool (69). The resulting output was then transferred to the online tool, NCBI Multiple Alignment. EV-D68 was selected as the anchor sequence, the top row, and BLOSUM62 was used for analysis and coloring (68). Amino acids identical to the anchor sequence (EV-D68) are represented by a dot and shown in blue, while amino acids not identical to the anchor sequence are shown in green a, with blue/green coloring indicating that the substitution is conservative.