Identification of diagnostic peptide regions that distinguish Zika virus from related mosquito-borne Flaviviruses

<div><p>Zika virus (ZIKV) is a member of the <i>Flavivirus</i> genus of positive-sense single-stranded RNA viruses, which includes Dengue, West Nile, Yellow Fever, and other mosquito-borne arboviruses. Infection by ZIKV can be difficult to distinguish from infection by other mosquito-borne Flaviviruses due to high sequence similarity, serum antibody cross-reactivity, and virus co-circulation in endemic areas. Indeed, existing serological methods are not able to consistently differentiate ZIKV from other Flaviviruses, which makes it extremely difficult to accurately calculate the incidence rate of Zika-associated Guillain-Barre in adults, microcephaly in newborns, or asymptomatic infections within a geographical area. In order to identify Zika-specific peptide regions that could be used as serology reagents, we have applied comparative genomics and protein structure analyses to identify amino acid residues that distinguish each of 10 <i>Flavivirus</i> species and subtypes from each other by calculating the specificity, sensitivity, and surface exposure of each residue in relevant target proteins. For ZIKV we identified 104 and 116 15-mer peptides in the E glycoprotein and NS1 non-structural protein, respectively, that contain multiple diagnostic sites and are located in surface-exposed regions in the tertiary protein structure. These sensitive, specific, and surface-exposed peptide regions should serve as useful reagents for seroprevalence studies to better distinguish between prior infections with any of these mosquito-borne Flaviviruses. The development of better detection methods and diagnostic tools will enable clinicians and public health workers to more accurately estimate the true incidence rate of asymptomatic infections, neurological syndromes, and birth defects associated with ZIKV infection.</p></div

Percentage of amino acid positions within surface diagnostic E protein peptides that overlap with known human B-cell Flavivirus epitopes.

<p>Percentage of amino acid positions within surface diagnostic E protein peptides that overlap with known human B-cell Flavivirus epitopes.</p

Comparison of the diagnostic peptide regions for all Flavivirus taxa infecting humans.

<p>Stacked bar chart of candidate diagnostic peptide sites across the E (upper panel) or NS1 (lower panel) for all 10 of the mosquito-borne Flavivirus species/subtypes evaluated in this study. Areas with darker shading indicate regions where all 10 taxa share a 15-mer peptide that is surface exposed and contains at least one diagnostic site.</p

Identification of diagnostic peptide regions in the NS1 non-structural protein for all Flavivirus taxa infecting humans.

<p>Stacked bar chart of candidate diagnostic sites (i.e. amino acid positions that were found to have a high degree of sensitivity and specificity for each Flavivirus taxon) within a sliding window (window size of 15, step size of 1) for the NS1 protein sequences in each of the Flavivirus taxa. Y-axis indicates the number of diagnostic residues (blue bars) or surface exposed residues (gray bars) in the 15-mer peptide starting at the protein amino acid position indicated on the x-axis. Surface-exposed diagnostic peptide regions containing at least 3 diagnostic sites and 6 solvent-accessible residues are represented with darker shading.</p

Identification of diagnostic peptide regions for ZIKV.

<p>Stacked bar chart of diagnostic sites having high sensitivity and specificity within a sliding window (window size of 15, step size of 1) for the ZIKV E (A) and ZIKV NS1 (B) proteins. Y-axis indicates the number of diagnostic residues (blue bars) or surface exposed residues (gray bars) in the 15-mer peptide starting at the protein amino acid position indicated on the x-axis. Surface-exposed diagnostic peptide regions containing at least 3 diagnostic sites and 6 solvent-accessible residues are represented with darker shading.</p

Location of a selected surface-exposed diagnostic peptide region in the E glycoprotein structure.

<p>(A) Plot providing higher resolution of the ZIKV E results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178199#pone.0178199.g001" target="_blank">Fig 1</a> with a red arrow indicating the surface diagnostic peptide highlighted in the lower panel. (B) Surface view of the structure for the ZIKV E:M heterodimer (PDB: 5IRE) is shown. The M chain is colored brown, the E chain is colored white, the selected 15-mer is colored red, residues that are surface exposed are colored blue, residues that overlap between the 15-mer peptide and surface exposed residues are colored purple, candidate diagnostic residues within the 15-mer that overlap with surface exposed residues are colored pink.</p

Summary statistics for the NS1 protein.

<p>Summary statistics for the NS1 protein.</p

NS1 protein sequence counts by species/subtype.

<p>NS1 protein sequence counts by species/subtype.</p

Summary statistics for the E protein.

<p>Summary statistics for the E protein.</p

Identification of diagnostic peptide regions in the E glycoprotein for all Flavivirus taxa infecting humans.

<p>Stacked bar chart of candidate diagnostic sites (i.e. amino acid positions that were found to have high sensitivity and specificity for each Flavivirus taxon) within a sliding window (window size of 15, step size of 1) for the E protein sequences in each of the Flavivirus taxa. Y-axis indicates the number of diagnostic residues (blue bars) or surface exposed residues (gray bars) in the 15-mer peptide starting at the protein amino acid position indicated on the x-axis. Surface-exposed diagnostic peptide regions containing at least 3 diagnostic sites and 6 solvent-accessible residues are represented with darker shading.</p