Diagnostic Test Divergence by Strain^a.

<p>Diagnostic Test Divergence by Strain<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161355#t002fn001" target="_blank">^a</a>.</p

ZIKV Phylogenomics.

<p>A Phylogenomic analysis based on the whole polyprotein sequence was generated with neighbor-joining methods. This tree was consistent with previously described isolate relationships based on NS5 and NS3 in resolving African and Asian lineages. The American strains form a distinct group most closely related to Asian strains.</p

Sequence Diversity Across ZIKV Strains.

<p>(A) Average nucleotide diversity (changes per 100 residues relative to consensus; grey bars) varied significantly (** <i>P</i><0.05 across strains). Average amino acid diversity changes per 100 residues relative to consensus; black bars) also varied significantly (* <i>P</i><0.05 across strains). (B) The ratio of amino acid changes per 100 residues to nucleotide changes per 100 residues also varied across strain. Individual domains within certain proteins are represented as indicated. Abbreviations used in this Fig are as follows: C-Capsid; PrP-Propeptide; gpM-Glycoprotein M; gp1-Glycoprotein 1; Glycoprotein C; Estm-E Stem; Pep-NS3 peptidase; DEAD-DEAD NTPase; Hel-Helicase; FtsJ-FtsJ domain; Poly-Polymerase; Clv Frg-Cleavage fragments.</p

N- and O-Linked Glycotypes of ZIKV Isolates.

<p>(A) N-linked glycosylation predictions (grey fill) for each strain (columns) were homologous across strains with the exception of E ASN₁₅₄ and NS2A ASN₁₄₈ (black fill). (B) O-linked glycosylation was highly variable across strains, with glycosylated residues represented by grey fill and non-glycosylated residues represented by black fill. Abbreviations used in this Fig are as follows: C-Capsid; PrP-Propeptide; gpM-Glycoprotein M; gp1-Glycoprotein 1; Glycoprotein C; Estm-E Stem; Pep-NS3 peptidase; DEAD-DEAD NTPase; Hel-Helicase; FtsJ-FtsJ domain; Poly-Polymerase; Clv Frg-Cleavage fragments.</p

Episodic Diversifying Selection in ZIKV.

<p>Individual proteins and/or functional domains varied in the proportion of amino acid sites under episodic diversifying selection. Many proteins or functional domains differed significantly (<i>P</i><0.05; boxed) from the background rate of selection derived from cleavage fragment sequences.</p

Nucleotide Conservation across Diagnostic Reagents.

<p>PCR primers and probes were aligned with 33 ZIKV genomes to explore their fidelity. Diagnostic reagents all had >10% divergence across residues (grey bars). The proportion of strains examined in this study that had at least one mismatch from diagnostic reagents ranged from from <10% to >95% (black bars).</p

Zika Virus Strain Information.

<p>Zika Virus Strain Information.</p

A Sialoreceptor Binding Motif in the <i>Mycoplasma synoviae</i> Adhesin VlhA

<div><p><i>Mycoplasma synoviae</i> depends on its adhesin VlhA to mediate cytadherence to sialylated host cell receptors. Allelic variants of VlhA arise through recombination between an assemblage of promoterless <i>vlhA</i> pseudogenes and a single transcription promoter site, creating lineages of <i>M. synoviae</i> that each express a different <i>vlhA</i> allele. The predicted full-length VlhA sequences adjacent to the promoter of nine lineages of <i>M. synoviae</i> varying in avidity of cytadherence were aligned with that of the reference strain MS53 and with a 60-a.a. hemagglutinating VlhA C-terminal fragment from a Tunisian lineage of strain WVU1853^T. Seven different sequence variants of an imperfectly conserved, single-copy, 12-a.a. candidate cytadherence motif were evident amid the flanking variable residues of the 11 total sequences examined. The motif was predicted to adopt a short hairpin structure in a low-complexity region near the C-terminus of VlhA. Biotinylated synthetic oligopeptides representing four selected variants of the 12-a.a. motif, with the whole synthesized 60-a.a. fragment as a positive control, differed (<i>P</i><0.01) in the extent they bound to chicken erythrocyte membranes. All bound to a greater extent (<i>P</i><0.01) than scrambled or irrelevant VlhA domain negative control peptides did. Experimentally introduced branched-chain amino acid (BCAA) substitutions Val3Ile and Leu7Ile did not significantly alter binding, whereas fold-destabilizing substitutions Thr4Gly and Ala9Gly tended to reduce it (<i>P</i><0.05). Binding was also reduced to background levels (<i>P</i><0.01) when the peptides were exposed to desialylated membranes, or were pre-saturated with free sialic acid before exposure to untreated membranes. From this evidence we conclude that the motif P-X-(BCAA)-X-F-X-(BCAA)-X-A-K-X-G binds sialic acid and likely mediates VlhA-dependent <i>M. synoviae</i> attachment to host cells. This conserved mechanism retains the potential for fine-scale rheostasis in binding avidity, which could be a general characteristic of pathogens that depend on analogous systems of antigenically variable adhesins. The motif may be useful to identify previously unrecognized adhesins.</p></div

PHM structural predictions.

<p>(<b>A</b>) The putative hemagglutination motif (PHM; red) was predicted to adopt a hairpin structure of two anti-parallel β strands separated by a short disordered loop. (<b>B</b>) The motif (red, indicated by arrow) mapped to a low-complexity region near the carboxyterminal domain (CTD) of the <i>M. synoviae</i> adhesin protein VlhA cleavage product MSPA, shown here in the structure predicted for the Tunisian lineage of strain WVU1853^T. The N-terminal domain (NTD) of MSPA was predicted to have much greater 3-dimensional complexity. (<b>C</b>) The length of the disordered loop was predicted to be longer in PHM peptides that bound to avian erythrocyte membranes (representing Florida and Tunisian lineages of strain WVU1853^T and strains FMT, K5016 and K5395) than in the reduced-binding peptide mutant FMT-Ala9Gly and the non-binding peptide representing strain MS53.</p

Erythrocyte membrane binding by PHM peptides.

<p>Bars depict mean ± standard error of the amount of synthetic peptide bound to avian erythrocyte membranes in an ELISA format (n = 3 independent replicates, with duplicate measurements of each peptide within replicate). The peptides represented variants of the putative hemagglutination motif (PHM) at the VlhA expression site of <i>M. synoviae</i> strains MS53, WVU1853^T (Florida and Tunisian lineages), FMT, K5016 and K5395, which spanned a>20-fold range in quantitative hemagglutination phenotypes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110360#pone.0110360-May1" target="_blank">[14]</a>. The positive control was the Tunisian lineage of strain WVU1853^T, and negative controls were scrambled strain FMT peptide and an irrelevant peptide from a distant site in VlhA from the Florida lineage of strain WVU1853^T. Different letters above the bar indicate means that differ (<i>P</i><0.05 or less) by Tukey-Kramer Honestly Significant Difference test. As predicted, the directed substitution Ala9Gly significantly reduced binding versus the parent peptide from strain FMT, and Thr4Gly tended to reduce binding, whereas Val3Ile and Leu7Ile did not significantly alter binding.</p