ImportanceNon-invasive techniques for retrieving ocular surface cells from babies infected by zika virus (ZIKV) during the gestational period remain to be determined.ObjectivesThe aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from Zika infected babies with and without Congenital Zika Syndrome (CZS) in satisfactory amount and quality to enable easy adoption in the field and application in the context of genomic and molecular approaches.Design, Settings, and ParticipantsOcular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. After an authorized written informed consent from the parents, samples were collected from both eyes of 12 babies (4 babies with maternal ZIKV exposure during gestation and presence of clinical signs which included ocular abnormalities and microcephaly; 4 babies with maternal ZIKV exposure during gestation but no clinical signs; and 4 unaffected control babies with negative PCR for Zika virus and without clinical signs). Cells were used for microscopy analyses and evaluated for their suitability for downstream molecular applications in transcriptomic and proteomic experiments.ResultsOur optimized impression cytology protocol enabled the capture of a considerable number of viable cells. The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed potential morphological alterations (cell keratinization, pyknosis, karyolysis, anucleation, and vacuolization). Molecular approaches were also performed in parallel. Genomic DNA and RNA were successfully isolated from all samples to enable the establishment of transcriptomic and proteomic studies.Conclusions and RelevanceOur method proved to be a suitable, fast, and non-invasive tool to obtain ocular cell preparations from babies with and without Zika infection. The method yielded sufficient cells for detailed morphological and molecular analyses of samples. We discuss perspectives for the application of impression cytology in the context of ZIKV studies in basic and clinical research.

IMPORTANCENoninvasive techniques for obtaining ocular surface cells (neuroepithelial) from babies with Congenital Zika Syndrome CZS - resulting from infection by zika virus (ZIKV) during gestational period (malformations include ocular abnormalities and microcephaly) - remain to be determined.OBJECTIVESThe aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from babies with CZS in satisfactory amounts and quality to enable the application in the context of genome approaches well as morphological and molecular evaluations.DESIGN, SETTINGS AND PARTICIPANTSIn this observational study, ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. Samples were collected with an authorized informed consent from both eyes of eight ZIKV infected babies according to the CZS diagnostic criteria (4 babies with positive PCR for Zika virus in gestation and presence of clinical signs which included ocular abnormalities and microcephaly and 4 babies with positive PCR for Zika virus during gestation but no clinical signs identified) and four unaffected babies (control samples / negative PCR, without clinical signs). Cells were used for microscopy analyses, transcriptomic and proteomic experiments and molecular procedure.MAIN OUTCOMES AND MEASURESThe microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures, including viability evaluation. In parallel, molecular approaches were performed.RESULTSOn impression cytology, a considerable amount of viable cells were captured. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed clear morphological alterations (cell keratinization, piknosis, karyolysis, anucleation and vacuolization). Genomic DNA and RNA were successfully isolated from all samples and allowed the establishment of transcriptomic and proteomic studies. Transcriptome analysis showed 8582 transcripts quantified in all samples and 63 differentially expressed genes in ocular cells from the exposed babies. Proteomics analysis allowed the identification of 2080, 2085 and 2086 high confident and unique proteins with at least one unique peptide in the unaffected, exposed to ZIKV and asymptomatic and CZS babies, respectively, being 2062 in common. Multivariate supervised analysis using the total quantitative protein features revealed a clear discrimination between the groups.CONCLUSIONS AND RELEVANCEOur method proved to be a suitable, fast, and non-invasive tool for detailed and precise morphological analyses with a perspective of application in OMIC studies for clinical and research studies of CZS.Key pointsQuestionAre the ocular surface cells of babies with Congenital Zika Syndrome viable to investigate the association between Zika virus infection during embryogenesis and ocular impairment?FindingsTo this date, this is the first study using an approach with perspectives in morphological, molecular and “OMICs” research from ocular samples captured by impression cytology of ZIKV infected babies during embryogenesis. The microscopic features of the conjunctival epithelial cells from all ZIKV infected babies showed clear morphological alterations.MeaningOcular cell surface capture offers a powerful model for studying the pathways involved in ocular diseases associated with ZIKV.

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In 2015, Brazil reported an outbreak identified as Zika virus (ZIKV) infection associated with congenital abnormalities. To date, a total of 86 countries and territories have described evidence of Zika infection and recently the appearance of the African ZIKV lineage in Brazil highlights the risk of a new epidemic. The spectrum of ZIKV infection-induced alterations at both cellular and molecular levels is not completely elucidated. Here, we present for the first time the gene expression responses associated with prenatal ZIKV infection from ocular cells. We applied a recently developed non-invasive method (impression cytology) which use eye cells as a model for ZIKV studies. The ocular profiling revealed significant differences between exposed and control groups, as well as a different pattern in ocular transcripts from Congenital Zika Syndrome (CZS) compared to ZIKV-exposed but asymptomatic infants. Our data showed pathways related to mismatch repair, cancer, and PI3K/AKT/mTOR signaling and genes probably causative or protective in the modulation of ZIKV infection. Ocular cells revealed the effects of ZIKV infection on primordial neuronal cell genes, evidenced by changes in genes associated with embryonic cells. The changes in gene expression support an association with the gestational period of the infection and provide evidence for the resulting clinical and ophthalmological pathologies. Additionally, the findings of cell death- and cancer-associated deregulated genes raise concerns about the early onset of other potential pathologies including the need for tumor surveillance. Our results thus provide direct evidence that infants exposed prenatally to the Zika virus, not only with CZS but also without clinical signs (asymptomatic) express cellular and molecular changes with potential clinical implications.

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