Molecular evolution and the global reemergence of enterovirus D68 by genome-wide analysis

Abstract

Human enterovirus D68 (EV-D68) was first reported in the United States in 1962; thereafter, a few cases were reported from 1970 to 2005, but 2 outbreaks occurred in the Philippines (2008) and the United States (2014). However, little is known regarding the molecular evolution of this globally reemerging virus due to a lack of whole-genome sequences and analyses. Here, all publically available sequences including 147 full and 1248 partial genomes from GenBank were collected and compared at the clade and subclade level; 11 whole genomes isolated in Taiwan (TW) in 2014 were also added to the database. Phylogenetic trees were constructed to identify a new subclade, B3, and represent clade circulations among strains. Nucleotide sequence identities of the VP1 gene were 94% to 95% based on a comparison of subclade B3 to B1 and B2 and 87% to 91% when comparing A, C, and D. The patterns of clade circulation need to be clarified to improve global monitoring of EV-D68, even though this virus showed lower diversity among clades compared with the common enterovirus EV-71. Notably, severe cases isolated from Taiwan and China in 2014 were found in subclade B3. One severe case from Taiwan occurred in a female patient with underlying angioimmunoblastic T-cell lymphoma, from whom a bronchoalveolar lavage specimen was obtained. Although host factors play a key role in disease severity, we cannot exclude the possibility that EV-D68 may trigger clinical symptoms or death. To further investigate the genetic diversity of EV-D68, we reported 34 amino acid (aa) polymorphisms identified by comparing subclade B3 to B1 and B2. Clade D strains had a 1-aa deletion and a 2-aa insertion in the VP1 gene, and 1 of our TW/2014 strains had a shorter deletion in the 5' untranslated region than a previously reported deletion. In summary, a new subclade, genetic indels, and polymorphisms in global strains were discovered elucidating evolutionary and epidemiological trends of EV-D68, and 11 genomes were added to the database. Virus variants may contribute to disease severity and clinical manifestations, and further studies are needed to investigate the associations between genetic diversity and clinical outcomes.

Figure 1

A maximum clade credibility tree by Bayesian phylogenetic analysis. A total of 169 EV-D68 strains were collected, including 147 complete and nearly complete genomes (including 11 TW strains from this study) and 22 TW strains in partial length, from 2007 to 2014. This phylogenetic tree based on the VP1 gene (933 nt long) presented 4 major clades, A to D. Clade B was further divided into B1 to B3. All TW/2014 and China/2014 strains were found in a new subclade, B3. The node label includes the name, country, and year of strains separated by vertical bars. The colors of the node and branch labels depend on their clades. A total of 84 US/2014, 4 Canada/2014, 1 Haiti/2014, and 1 US/2013 strains in subclade B1 are represented by a triangle; their sequence counts are shown in parentheses. EV-D68 = enterovirus D68.

Figure 2

The 34 amino acid polymorphisms of clade B. Thirty-four amino acid polymorphisms were identified from EV-D68 genomes in a length of approximately 2190 aa, including 7 in VP1, 6 in 3D, 5 in 2A, 5 in VP2, 3 in 3C, 2 each in VP4, VP3, and 3A, and 1 each in 2B and 3C. Five positions (i.e., V18, T207, A/V220, T470, and G558) were newly reported by comparing subclade B3 with B1 and B2; these positions are marked in red. A previous study reported 6 polymorphisms (i.e., M291, V341, T860, D927, S1108, and R2005 in subclade B3), which are marked with a superscript asterisk. Aa = amino acid, EV-D68 = enterovirus D68.

Figure 3

The 29 nonconserved residues of 11 Taiwan/2014 genomes. Twenty-nine nonconserved residues were identified, including 7 in VP3, 5 in VP1, 4 in 2C, 3 each in VP2, 2A, 2B, and 3D, and 1 in 3A. These residues in the same aligned column were identical to the first sequence TW-02795-2014 and are masked by dots. Position 474 was the most divergent site, with 7 F's and 4 L's, followed by positions 558, 868, 1031, 1141, and 1190 with 3 substitutions, positions 377, 551, 849, and 2161 with 2 substitutions, and another 19 sites with only 1 substitution. Positions 558, 868, 1031, 1141, and 1190 are marked in red; they exhibit residues “G,” “A,” “S,” “T,” and “F” in 3 Linkou CGMH cases, but “A,” “V,” “N,” “N,” and “Y” in the other 8 TW cases and subclades B1 and B2, except for position 1141 in subclade B1. CDC = Centers for Disease Control and Prevention, CGMH = Chang Gung Memorial Hospital.