Postnatal Identification of Zika Virus Peptides from Saliva.

We explored the potential to diagnose Zika virus (ZIKV) infection by analyzing peptides in saliva during a convalescent phase of infection, long after resolution of acute disease. A 25-y-old woman clinically diagnosed with Zika fever in the first trimester was enrolled with her dizygotic twins for a 3-mo postnatal sample of saliva (9-mo after maternal infection). The female baby (A) had microcephaly while the male baby (B) was born healthy. Peptidomic analysis was completed by mass spectrometry (MS/MS), and ZIKV peptides were identified using the National Institutes of Health Zika Virus Resource database, then aligned and mapped to the ZIKV polyprotein to determine proteome coverage and phylogenetic studies. A total of 423 (mother), 607 (baby A), and 183 (baby B) unique ZIKV peptides were identified in saliva by MS/MS, providing a coverage of 67%, 84%, and 45%, respectively, of the entire ZIKV polyprotein (>3,400 amino acids). All peptides were aligned to other flaviviruses that are circulating in Brazil (dengue and yellow fever) to discard false-positive matches. Nine peptides identified were highly conserved to dengue virus. Alignment of a contiguous peptide sequence for mother/babies with the 74 ZIKV sequences suggested that the virus may have entered the oral cavity through the salivary glands, leading to an infection that persists into the postnatal period (vertical transmission). Furthermore, we identified 9 sequence variations that were unique to the baby with microcephaly (not found in the mother or the twin). This sequence information could provide a template for future neuropathogenic studies. A much larger sample size is required to determine whether sequence variation in the envelope protein significantly associates with microcephaly. Finally, from a public health perspective, it will be important to determine whether viral replication is still taking place after birth and whether the virus can be transmitted through salivary contact.

Duplicated or Hybridized Peptide Functional Domains Promote Oral Homeostasis.

Proteins that have existed for millions of years frequently contain repeats of functional domains within their primary structure, thereby improving their functional capacity. In the evolutionary young statherin protein contained within the in vivo-acquired enamel pellicle (AEP), we identified a single functional domain (DR9) located within the protein N-terminal portion that exhibits a higher affinity for hydroxyapatite and more efficient protection against enamel demineralization compared to other native statherin peptides. Thus, we tested the hypothesis that multiplication of functional domains of naturally occurring pellicle peptides amplifies protection against enamel demineralization. In addition, a specific amino acid sequence from histatin 3 (RR-14) was introduced to the hybrid peptides for further testing. Enamel specimens were sectioned to 150-µm thickness and randomly grouped as follows: DR9, DR9-DR9, DR9-RR14, statherin, histatin 1, or distilled water (control). After submersion for 2 h at 37°C, the specimens were placed in 2 mL demineralization solution for 12 d at 37°C. Upon sample removal, the remaining solution was subjected to colorimetric assays to determine the amount of calcium and phosphate released from each specimen. DR9-DR9 amplified protection against enamel demineralization when compared to single DR9 or statherin. Notably, the hybrid peptide DR9-RR14 demonstrated relatively strong protection when the antimicrobial property of these peptides was tested against Candida albicans and Streptococcus mutans. DR9-RR14 was able to maintain 50% of the antifungal activity compared with RR14 for C. albicans and similar values of S. mutans killing activity. This study has pioneered the functional exploration of the natural peptide constituents of the AEP and their evolution-inspired engineered peptides. The knowledge obtained here may provide a basis for the development of stable (proteinase-resistant) synthetic peptides for therapeutic use against dental caries, dental erosion, and/or oral candidiasis.