Specific residue variations detected in the EVD68 VP1 protein from the 2014 outbreak strains.

<p><b>(A)</b> VP1 protein alignment based on the Clade B subcluster. The alignment was performed with MEGA5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.ref036" target="_blank">36</a>]. Hyphens indicate residues identical to those in CA/AFP/11-1767; “X” indicates unidentified amino acid due to unidentified nucleotides in the submitted CA/AFP/v12T04950 sequence. Numbers indicate protein positions. Sequence CQ5585 is missing the fragment surrounding position 290. The subtree for the subclustered Clade B strains is extracted from the tree in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.g001" target="_blank">Fig 1</a>. (<b>B)</b> Structures of viral proteins VP1 (green), VP2 (purple), and VP3 (cyan) in the surface model. The remodeling was based on the published EVD68 structure (PDB: 4WM8) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.ref020" target="_blank">20</a>]. Positions 218 and 290, located on the surface of VP1, are labeled in red. Position 99 is shown in red instead of 98 since the latter is missing in the published EVD68 structure. (<b>C)</b> Position 194, is located beneath the viral surface and is essential for β-sheet formation, which may be important for supporting EVD68 virion “canyon” formation. Left panel: the surface model of VP1 rotated 120° from the model shown in Fig 2B; right panel: a cartoon model of the β-sheet in which residue 194 interacts with residue 183.</p

Host-receptor binding and/or viral antigenicity correlate with EVD68 clade classification.

<p><b>(A).</b> Surface models of EVD68 VP1 (green), VP2 (purple), and VP3 (cyan), with clade-specific residues labeled in red. The remodeling was based on the published EVD68 structure (PDB: 4WM8) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.ref020" target="_blank">20</a>]. Positions 140 and 149 are labeled in red to indicate the approximate positions of residues 141, 143, 144, 145, and 148 that are missing in the published structure. <b>(B).</b> Structural alignment of VP1 from the published EVD68 structure (green) or the proposed structure (blue) based on the viral structure of Echovirus 7 (PDB: 2X5I) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.ref038" target="_blank">38</a>]. The alignment was performed with PyMol. (<b>C).</b> Surface model of the proposed EVD68 VP1 structure (blue), with clade-specific residues indicated in red. The panel on the right provides a detailed cartoon model showing clade-specific residues (in red) located on the loops forming the “canyon” responsible for host-receptor interaction. The BC-loop and DE-loop, thought to be critical in both host-receptor binding and viral antigenicity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.ref040" target="_blank">40</a>], are labeled with orange frames.</p

Evolutionary selection on the 18 clade-specific positions.

<p>After sequence alignment, the dN-dS values for each clade were calculated for these clade-specific positions with the help of MEGA5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.ref036" target="_blank">36</a>]. Values >0 indicate positive selection, while values <0 indicate purifying selection. Each codon with dN-dS = 0 in this study shared conserved nucleotides and thus was considered as purifyingly selected. Clade D was excluded from this analysis because only two strains contained full-length VP1.</p

Amino acid variation in the VP1 residues of different EV68 clades.

<p>The comparison was performed with the full-length EV68 VP1 region (306 amino acids) from 117 EVD68 sequences. The clade classification is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.g001" target="_blank">Fig 1</a>. Values in the lower-left indicate the average number of different amino acids between the clades, with the range in parentheses. Values in the upper-right indicate the relative percentage of the VP1 region that differs among the clades. Sequence alignment and calculation were performed using MEGA5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.ref036" target="_blank">36</a>].</p><p>Amino acid variation in the VP1 residues of different EV68 clades.</p

Phylogenetic analysis of full-length VP1 region of EVD68 strains.

<p>117 full-length EVD68 VP1 sequences were used for the re-construction of the Neighbor-Joining tree (<b>A</b>). Similar clade formation was detected compared to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.g001" target="_blank">Fig 1</a>, and each clade was shown as a subtree (<b>B, C, and D,</b> for Clade A, B, and C respectively). The tree was re-constructed with the help of MEGA5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144208#pone.0144208.ref036" target="_blank">36</a>]. Only bootstrap values >70 were shown. For a clearer presentation of the relationship between strains, only the topology was shown. Circle symbols indicate EVD68 strain from the 2014 outbreak, while square symbols indicate compressed EVD68 clades.</p

Circulating HFMD-Associated Coxsackievirus A16 Is Genetically and Phenotypically Distinct from the Prototype CV-A16

<div><p>Human enteroviruses (HEV) have been linked to hand, foot, and mouth disease (HFMD) in the Pacific and Southeast Asia for decades. Many cases of HFMD have been attributed to coxsackievirus A16 (CV-A16, CA16), based on only partial viral genome determination. Viral phenotypes are also poorly defined. Herein, we have genetically and phenotypically characterized multiple circulating CV-A16 viruses from HFMD patients and determined multiple full-length sequences of these circulating viruses. We discovered that the circulating CV-A16 viruses from HFMD patients are genetically distinct from the proto-type CV-A16 G10. We have also isolated circulating CV-A16 viruses from hospitalized HFMD patients and compared their virological differences. Interestingly, circulating CV-A16 viruses are more pathogenic in a neonatal mouse model than is CV-A16 G10. Thus, we have found circulating recombinant forms of CV-A16 (CRF CV-A16) that are related to, but different from, the prototype CV-A16 G10 that have distinct biological phenotypes.</p></div

Summary of Changchun024 recombination events detected by RDP4.

<p>Summary of Changchun024 recombination events detected by RDP4.</p

Phylogenetic analysis of eight CV-A16 full-length genomic sequences isolated from HFMD patients in Changchun, China.

<p>(A)The complete genomic sequences of 3 other CV-A16 strains from China and all of the 21 HEV reference sequences were retrieved from Genbank. Phylogenetic analysis was conducted using MEGA4 software employing the neighbor-joining method with 1,000 replications and the Kimura 2-parameter model. Bootstrap values greater than 70% are shown. The ▪icon indicates CV-A16 strains isolated from Changchun; the ♦icon indicates the prototype CV-A16-G10. (B) and (C) Identification of recombinant circulating CV-A16 strains of Changchun024 and Changchun029 by bootscanning. (B) Bootscanning analysis of Changchun024 as the query sequence. (C) Bootscanning analysis of Changchun029 as the query sequence. For all HEV-A sequences together with other two outgroups EV68 and poliovirus 1, Changchun024 and Changchun029 showed possible recombination with CA4, CV-A16-G10, and EV71A. Bootscanning was generated with Simplot 3.5.1 software using a sliding window size of 500 bases and step size of 20 bases at a time. The <i>y</i> axis shows the percentage of the permuted tree in which the selected HEV virus sequences cluster with the query sequence.</p

Clinical features of eight HFMD patients infected with CV-A16 viruses from Changchun.

<p>Clinical features of eight HFMD patients infected with CV-A16 viruses from Changchun.</p

Disease and mortality rate differences in newborn mice caused by different strains of CV-A16.

<p>Differences in virulence between the prototype CV-A16-G10 and circulating recombinant forms of CV-A16 in neonatal mice were compared. One-day-old ICR mice were intracerebrally inoculated with 10^3.5 TCID50/ml G10, Changchun024, Changchun045, Changchun090 viruses in DMEM medium. The negative control mice were given medium instead of the virus suspension. The survival rates (A) and clinical scores (B) were monitored and recorded daily after infection for 21 days. The clinical score was graded as follows: 0: no abnormalities; 1: lethargy and inactivity; 2: wasting; 3: limb shake weakness; 4: hind-limb paralysis; 5: moribund or dead. Each group contained six to ten mice.</p