Journal of Virology
J Virol. 2014 Mar;88(5):2374-84. doi: 10.1128/JVI.03070-13. Epub 2013 Dec 26.
Antigenic and Receptor Binding Properties of Enterovirus 68
Tadatsugu Imamuraa,
Michiko Okamotoa,
Shin-ichi Nakakitab,
Akira Suzukia,
Mariko Saitoc,
Raita Tamakia,
Socorro Lupisang,
Chandra Nath Roya,
Hiroaki Hiramatsuf,
Kan-etsu Sugawarad,
Katsumi Mizutae,
Yoko Matsuzakid,
Yasuo Suzukif and
Hitoshi Oshitania
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.
Human enterovirus 68 (EV68) (species, Human enterovirus D; genus, Enterovirus; family, Picornaviridae) was first isolated in the United States from pediatric patients hospitalized with lower-respiratory-tract infections in 1962 (
1).
Since its first detection, EV68 has only rarely been identified. In an enterovirus surveillance conducted in the United States from 1970 to 2005, only a total of 26 strains were reported over 35 years (
2).
However, the number of reported EV68 cases increased dramatically in the late 2000s, in different parts of the world (
3–8). The underlying molecular mechanism for this sudden increase of EV68 detections in recent years is still unknown
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Considering that all EV68 strains tested showed binding specificity to α2,6-linked sialic acid terminals, EV68 might have affinity for the upper respiratory tract. However, several previous studies reported that the detection rate of EV68 was significantly higher among patients hospitalized with severe lower-respiratory-tract infections than those visiting outpatient clinics with mild upper-respiratory-tract infections (
6,
8,
10). Therefore, there might be unknown mechanisms for severe infections with EV68 other than the distribution of viral receptors.
In the species human enterovirus D (HEV-D), three serotypes, including EV68, EV70, and EV94 are known to causes illness in human (
31,
32). The VP1 sequences, the most variable genome region of enteroviruses, EV68 were shown to be closely related to those of EV70 and EV94 (
26,
32). It was previously reported that EV70 has affinity for α2-3 SAs (
33). Although the genome regions of EV68 and EV70 associated with their affinity for SAs are still unknown, it might be possible that EV68 and EV70 share similar amino acid sequences, which are responsible for the receptor binding.
It is well known that influenza viruses have neuraminidase (sialidase) activity, by which the newly synthesized viral proteins are removed from SAs, and the virus progenies are released from the host cell surface (
34). However, in our study, we did not detect such enzymatic activities for EV68 by the MUNANA assay. Several members of the family Picornaviridae are reported to have affinity for SAs (
33,
35); however, it is unknown if they have sialidase activities. It was previously shown that, in contrast to enveloped viruses, nonenveloped viruses are released from the host cells by disintegration of the cells by using mechanisms such as cell lysis (
36). On the other hand, influenza viruses are enveloped viruses, and the budding of the viral progenies is dependent on the interaction between the host cell membrane and the viral proteins, including HA and NA (
34). The reasons for the lack of sialidase activity in EV68 strains might lie in those differences in the virus-release mechanisms.
In conclusion, our study showed that EV68 detected in recent outbreaks possesses highly divergent antigenicity compared to strain Fermon. The emergence of strains with different antigenicity was suggested as a possible mechanism for the recent increase in the rate of detection. However, further studies using strains detected at different time points between 1962 and 2010 are required for drawing any conclusions. Moreover, the antigenic differences were also detected among the strains of 3 genetic groups. The roles of those antigenic differences in the transmission dynamics of EV68 in the community also need to be further clarified.
We also demonstrated that EV68 preferably binds to α2-6 SAs without sialidase activities. In the study, we did not find any differences in the receptor binding properties among the EV68 strains; therefore, the association of the receptor recognition patterns with the recent EV68 outbreaks remains elusive. Furthermore, although our findings suggested that EV68 receptors are commonly distributed in the upper respiratory tract in humans, there is epidemiological and clinical evidence suggesting that EV68 is more likely to cause severe respiratory infections. Therefore, further studies are needed to gain an overview of pathogenesis for the severe respiratory illnesses associated with EV68 infections.
Interesting since enterovirus EV 71 is the usually associated with poliomyelitis-like paralysis
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