Distinct genetic clades of enterovirus D68 detected in 2010, 2013, and 2015 in Osaka City, Japan

<div><p>The first upsurge of enterovirus D68 (EV-D68), a causative agent of acute respiratory infections (ARIs), in Japan was reported in Osaka City in 2010. In this study, which began in 2010, we surveyed EV-D68 in children with ARIs and analyzed sequences of EV-D68 strains detected. Real-time PCR of 19 respiratory viruses or subtypes of viruses, including enterovirus, was performed on 2,215 specimens from ARI patients (<10 years of age) collected between November 2010 and December 2015 in Osaka City, Japan. EV-D68 was identified in 18 enterovirus-positive specimens (<i>n</i> = 4 in 2013, <i>n</i> = 1 in 2014, and <i>n</i> = 13 in 2015) by analysis of viral protein 1 (VP1) or VP4 sequences, followed by a BLAST search for similar sequences. All EV-D68 strains were detected between June and October (summer to autumn), except for one strain detected in 2014. A phylogenetic analysis of available VP1 sequences revealed that the Osaka strains detected in 2010, 2013, and 2015 belonged to distinct clusters (Clades C, A, and B [Subclade B3], respectively). Comparison of the 5′ untranslated regions of these viruses showed that Osaka strains in Clades A, B (Subclade B3), and C commonly had deletions at nucleotide positions 681–703 corresponding to the prototype Fermon strain. Clades B and C had deletions from nucleotide positions 713–724. Since the EV-D68 epidemic in 2010, EV-D68 re-emerged in Osaka City, Japan, in 2013 and 2015. Results of this study indicate that distinct clades of EV-D68 contributed to re-emergences of this virus in 2010, 2013, and 2015 in this limited region.</p></div

Alignment of nucleotide sequences of the 5′ UTR of strains in the three major genetic clades of EV-D68.

<p>Nucleotide sequences (nucleotide positions 501–1,000, corresponding to those of the Fermon strain) were aligned in MEGA 7.0. A partial sequence (nucleotide positions 671–740 of Fermon strain) is shown. Hyphens denote deleted nucleotides.</p

Alignment of nucleotide sequences of the 5′ UTR of strains in the three major genetic clades of EV-D68.

<p>Nucleotide sequences (nucleotide positions 501–1,000, corresponding to those of the Fermon strain) were aligned in MEGA 7.0. A partial sequence (nucleotide positions 671–740 of Fermon strain) is shown. Hyphens denote deleted nucleotides.</p

Phylogeny of complete VP1 gene sequences.

<p>Complete VP1 gene sequences (927 nt, corresponding to nucleotide positions 2,389–3,315 of the Fermon prototype strain of EV-D68) were analyzed. The phylogenetic tree was constructed and evaluated with 1,000 bootstrap pseudoreplicates using MEGA 7.0 software. Based on the Akaike information criterion with correction for finite sample sizes, a general time reversible (GTR) plus gamma distributed with invariant sites (G+I) model was used. Numbers at nodes, which indicate bootstrap support values (>85%), are shown. Sequences in GenBank were also included in the analysis. Strain name, country of origin, and year of detection are shown for each strain. GenBank accession numbers are presented in parentheses. The scale bar shows the genetic distance.</p

Phylogenetic analysis of complete or near-complete genome sequences of EV-D68 strains.

<p>Complete or near-complete genome sequences of EV-D68 were used to construct a maximum likelihood phylogram. The phylogenetic tree was constructed and evaluated with 1,000 bootstrap pseudoreplicates using MEGA 7.0 software. Based on the Akaike information criterion with correction for finite sample sizes, a GTR plus G+I model was used. Numbers at nodes, which indicate bootstrap support values (>95%), are given. Sequences in GenBank were also included in the analysis. Strain name, country of origin, and year of detection are shown for each strain. GenBank accession numbers are presented in parentheses. The scale bar shows the genetic distance.</p

Distribution of EV-D68 in Osaka City, Japan, from January 2010 to December 2015.

<p>The unbroken line represents the number of specimens collected per month. The shaded bar shows EV-D68-positive specimens from patients with ARIs. Our previous data from January to October 2010 are also shown.</p

Comparison of VP1 region amino acid sequences between EV-D68 strains in distinct genetic clades.

<p>An alignment of partial amino acid sequences of the EV-D68 strains was analyzed. Strains in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184335#pone.0184335.g003" target="_blank">Fig 3</a> were used in this analysis.</p

VP1 amino acid and nucleotide sequence identities between subclade B3 strains from Osaka and worldwide.

<p>VP1 amino acid and nucleotide sequence identities between subclade B3 strains from Osaka and worldwide.</p

Information on EV-D68-positive patients and EV-D68 detected in 2010–2015 in Osaka City, Japan.

<p>Information on EV-D68-positive patients and EV-D68 detected in 2010–2015 in Osaka City, Japan.</p