Antigenic and receptor binding properties of enterovirus 68

Abstract

Increased detection of enterovirus 68 (EV68) among patients with acute respiratory infections has been reported from different parts of the world in the late 2000s since its first detection in pediatric patients with lower-respiratory-tract infections in 1962. However, the underlying molecular mechanisms for this trend are still unknown. We therefore aimed to study the antigenicity and receptor binding properties of EV68 detected in recent years in comparison to the prototype strain of EV68, the Fermon strain. We first performed neutralization (NT) and hemagglutination inhibition (HI) tests using antisera generated for EV68 strains detected in recent years. We found that the Fermon strain had lower HI and NT titers than recently detected EV68 strains. The HI and NT titers were also significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis. In glycan array analysis, all tested EV68 strains showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study demonstrates that emergence of strains with different antigenicity is the possible reason for the increased detection of EV68 in recent years. Additionally, we found that EV68 preferably binds to α2-6 SAs, which suggests that EV68 might have affinity for the upper respiratory tract.

Importance: Numbers of cases of enterovirus 68 (EV68) infection in different parts of the world increased significantly in the late 2000s. We studied the antigenicity and receptor binding properties of recently detected EV68 strains in comparison to the prototype strain of EV68, Fermon. The hemagglutination inhibition (HI) and neutralization (NT) titers were significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis, which showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study suggested that the emergence of strains with different antigenicities was the possible reason for the increased detections of EV68 in recent years. Additionally, we revealed that EV68 preferably binds to α2-6 SAs. This is the first report describing the properties of EV68 receptor binding to the specific types of sialic acids.

FIG 1

NT and HI titers of EV68 strains against antisera generated for EV68 detected in recent years. The circles indicate the mean HI titers (a to c) and NT titers (d to f) among strains of each lineage, and whiskers indicate the 95% confidence intervals for the mean values. The P value for the difference of titers between lineages are indicated above the brackets. Samples were tested in duplicate in HI tests and quadruplicate in NT tests, and each experiment was performed separately 2 to 4 times.

FIG 2

Receptor binding specificities of EV68. The receptor binding specificities of 12 strains of EV68 was measured for 6 types of sialyloligosaccharides by glycan array analysis. Samples were tested in triplicate, and three separate experiments were performed. The fluorescent intensity for each glycan was calculated as the average of 3 determinations.

FIG 3

Sialidase activity of EV68 for the MUNANA substrate. The sialidase activities of four strains of EV68 for MUNANA were measured with 5 different HA levels. One strain of influenza A virus, (H1N1) pdm, was used as a positive control. Samples were tested in triplicate, and three separate experiments were performed. Error bars indicate standard errors of the mean.

FIG 4

Complete VP1, VP2, and VP3 amino acid sequences of EV68 strains used for analysis. The complete amino acid sequences of the regions VP2 (a), VP3 (b), and VP1 (c) of EV68 strains used for analysis were aligned with that the prototype strain, Fermon (AF081348). The positions where the sequences of strains used for this analysis had amino acid residues identical to those of strain Fermon are indicated with dots. The BC and DE loop regions in VP1 are boxed.

FIG 5

Codons under positive selection in region VP1. Selection analysis was conducted for 3 sequence data sets, including EV68 strains of lineages 1, 2, and 3, by the SLAC method. Codons with dN/dS values higher than 0 were defined as being under positive selection. The amino acid sequences of the BC and DE loop regions are boxed.