Abstract

   During recent Zika epidemics, adults infected with Zika virus (ZIKV)
   have developed organ-specific inflammatory complications. The most
   serious Zika-associated inflammatory eye disease is uveitis, which is
   commonly anterior in type, affecting both eyes and responding to
   corticosteroid eye drops. Mechanisms of Zika-associated anterior
   uveitis are unknown, but ZIKV has been identified in the aqueous humor
   of affected individuals. The iris pigment epithelium is a target cell
   population in viral anterior uveitis, and it acts to maintain immune
   privilege within the anterior eye. Interactions between ZIKV and human
   iris pigment epithelial cells were investigated with infectivity assays
   and RNA-sequencing. Primary cell isolates were prepared from eyes of 20
   cadaveric donors, and infected for 24 hours with PRVABC59 strain ZIKV
   or incubated uninfected as control. Cytoimmunofluorescence, RT-qPCR on
   total cellular RNA, and focus-forming assays of culture supernatant
   showed cell isolates were permissive to infection, and supported
   replication and release of infectious ZIKV. To explore molecular
   responses of cell isolates to ZIKV infection at the whole transcriptome
   level, RNA was sequenced on the Illumina NextSeq 500 platform, and
   results were aligned to the human GRCh38 genome. Multidimensional
   scaling showed clear separation between transcriptomes of infected and
   uninfected cell isolates. Differential expression analysis indicated a
   vigorous molecular response of the cell to ZIKV: 7,935 genes were
   differentially expressed between ZIKV-infected and uninfected cells
   (FDR < 0.05), and 99% of 613 genes that changed at least two-fold were
   up-regulated. Reactome and KEGG pathway and Gene Ontology enrichment
   analyses indicated strong activation of viral recognition and defense,
   in addition to biosynthesis processes. A CHAT network included 6275
   molecular nodes and 24 contextual hubs in the cell response to ZIKV
   infection. Receptor-interacting serine/threonine kinase 1 (RIPK1) was
   the most significantly connected contextual hub. Correlation of gene
   expression with read counts assigned to the ZIKV genome identified a
   negative correlation between interferon signaling and viral load across
   isolates. This work represents the first investigation of mechanisms of
   Zika-associated anterior uveitis using an in vitro human cell model.
   The results suggest the iris pigment epithelium mounts a molecular
   response that limits intraocular pathology in most individuals.

   Keywords: Zika, virus, ZIKV, human, eye, iris, epithelium, pigment
   epithelial cell

Introduction

   Zika virus (ZIKV) is a single-stranded RNA flavivirus, which was first
   isolated from non-human primates in Uganda in 1947, and from humans in
   the same country in the early 1950s ([47]1). Zika virus disease is
   generally considered to pose little risk to human health unless it is
   contracted in utero. In recent epidemics from 2013 to 2014 in French
   Polynesia, and from 2015 to 2016 in Brazil and other American nations,
   women who were infected while pregnant delivered infants with a
   spectrum of developmental abnormalities ([48]2, [49]3), now referred to
   as congenital Zika syndrome ([50]4). Characteristic abnormalities in
   this disease include severe microcephaly, thinned cerebral cortex with
   subcortical calcification, extrapyramidal motor disturbances and
   contractures. In addition, approximately 70% of babies with congenital
   Zika syndrome develop ocular abnormalities, including retinal
   dystrophic lesions that show a preference for the macula, and therefore
   cause blindness ([51]5). However, in some adults, infection with ZIKV
   results in serious inflammatory conditions ([52]1).

   Uveitis –or intraocular inflammation– represents a diverse spectrum of
   diseases, classified according to anatomical localization within the
   eye and by specific phenotype ([53]6). Different types of uveitis have
   been reported in adult patients infected with ZIKV ([54]7). The most
   common form is anterior uveitis –based in the iris and ciliary body–
   which occurs in approximately 50% of patients who present with “red
   eyes” in the context of Zika fever ([55]8). This uveitis is typically
   bilateral, may impact the visual acuity, and often is accompanied by an
   elevation of intraocular pressure, as we ([56]9) and others ([57]8,
   [58]10, [59]11) have documented ([60] Table 1 ). In one of the first
   reports of Zika-associated anterior uveitis, aqueous humor was
   collected from the eye of a patient treated with corticosteroid eye
   drops alone; ZIKV RNA was recovered from fluid taken at 8 days after
   the onset of systemic symptoms, but not at 16 days ([61]9). This
   implies that Zika-associated anterior uveitis is associated with the
   presence of ZIKV inside the eye.

Table 1.

   Clinical features of ZIKV-associated anterior uveitis diagnosed in
   patients presenting to the Ophthalmology Clinic of Ribeirão Preto
   General Hospital, Ribeirão Preto, São Paulo or reported in the medical
   literature.
   Location; Demographics (Reference if applicable)* Clinical features
   Basis for diagnosis Treatment and course
   Ribeirão Preto, Brazil;
   M, 40 years ([62]9) OD/OS; VA=20/20 OD, 20/60 OS; AC cell=0.5+ OD, 2+
   OS; IOP=16mmHg OD, 15mmHg OS; other=punctate keratitis Epidemiology;
   Systemic features; Serology; Aqueous and blood RT-PCR Topical
   glucocorticoid; Remission of uveitis with improvement of VA
   Ribeirão Preto, Brazil;
   F, 55 years OD/OS; VA=20/30 OD, 20/25 OS; AC cell=3+ OD, 2+ OS;
   IOP=34mmHg OD, 17mmHg OS Epidemiology; Systemic features; Aqueous and
   blood RT-PCR Topical glucocorticoid and hypotensive; Remission of
   uveitis with improvement of VA
   Ribeirão Preto, Brazil;
   F, 41 years OD; VA=20/100 OD, 20/20 OS (unrelated corneal dystrophy
   OD); AC cell=3+ OD, 0+ OS; IOP=17mmHg OD, 16mmHg OS Epidemiology;
   Systemic features; Negative work-up for other causes Topical
   glucocorticoid; Remission of uveitis with no change in VA
   Ribeirão Preto, Brazil;
   F, 40 years OS; VA=20/100 OD, 20/30 OS (unrelated toxoplasmic retinal
   scar OD); AC cell=0+ OD, 1+ OS; IOP=16mmHg OD, 16mmHg OS Epidemiology;
   Systemic features; Negative work-up for other causes Topical
   glucocorticoid; Remission of uveitis with improvement of VA
   Ribeirão Preto, Brazil;
   F, 66 years OD; VA=20/40 OD, 20/30 OS; (unrelated macular hole OD); AC
   cell=2+ OD, 0+ OS; IOP=14mmHg OD, 14mmHg OS Epidemiology; Systemic
   features; Negative work-up for other causes Topical glucocorticoid;
   Remission of uveitis with no change in VA
   Rio de Janeiro, Brazil;
   M, 39 years ([63]10) OD/OS; VA=20/40 OD, 20/40 OS; AC cell=1+ OD, 1+
   OS; IOP=40mmHg OD, 28mmHg OS Epidemiology; Systemic features; Negative
   work-up for other causes Topical glucocorticoid and hypotensive;
   Remission of uveitis with improvement of VA and lowering of IOP
   Martinique, France;
   W, 50s ([64]11) OD/OS; VA=20/25 OD, 20/20 OS; AC cell present (grade
   NS); IOP=36mmHg OD, 26mmHg OS Epidemiology; Systemic features; Urine
   RT-PCR; Negative work-up for other causes Topical hypotensive;
   Remission of uveitis and lowering of IOP#
   Martinique, France;
   W, 60s ([65]11) OD/OS; VA=20/40 OD, 20/20 OS; AC cell present (grade
   NS); IOP=58mmHg OD, 32mmHg OS; other=corneal edema Epidemiology;
   Systemic features; Aqueous and urine RT-PCR; Negative work-up for other
   causes Topical hypotensive; Remission of uveitis and lowering of IOP
   Guadeloupe, France;
   F, adult (age NS) ([66]8) OD/OS; VA=20/20 OD, 20/25 OS; AC cell=0.5+
   OD, 1+ OS;
   IOP normal (value NS) Epidemiology; Systemic features; Aqueous RT-PCR;
   Negative work-up for other causes Topical glucocorticoid; Remission of
   uveitis and recovery of VA
   Guadeloupe, France;
   M, adult (age NS) ([67]8) OD/OS; VA=20/20 OD, 20/25 OS; AC cell=0 OD,
   0.5+ OS; IOP=16mmHg OD, 28mmHg OS Epidemiology; Systemic features;
   Aqueous RT-PCR; Negative work-up for other causes Topical
   glucocorticoid and hypotensive; Remission of uveitis; Persistent
   elevation of IOP
   Guadeloupe, France;
   Gender NS, adult (Age NS) ([68]8) OD/OS; VA=20/32 OD, 20/20 OS; AC
   cell=0 OD, 1+ OS; IOP=25mmHg OD, 16mmHg OS Epidemiology; Systemic
   features; Aqueous RT-PCR; Negative work-up for other causes Topical
   glucocorticoid and hypotensive; Remission of uveitis; Persistent
   elevation of IOP
   Guadeloupe, France;
   Gender NS, adult (Age NS) ([69]8) OD/OS; VA=20/20 OD, 20/40 OS; AC
   cell=0 OD, 1+ OS; IOP=16mmHg OD, 30mmHg OS Epidemiology; Systemic
   features; Aqueous RT-PCR; Negative work-up for other causes Topical
   glucocorticoid and hypotensive; Remission of uveitis; Persistent
   elevation of IOP
   [70]Open in a new tab

   AC, anterior chamber; F, female; IOP, intraocular pressure; M, male;
   NS, not specified; OD, right eye; OS, left eye; RT-PCR, reverse
   transcription-polymerase reaction; VA, visual acuity.

   *Reference 8 reports an observational study of 62 patients that
   included case histories from 4 patients whose diagnoses were based on
   aqueous RT-PCR plus other tests for Zika. Results of other tests were
   not specified. Visual acuity outcome was not stated for 3 of the 4
   cases.

   Several independently conducted studies have explored the pathogenic
   mechanisms of congenital Zika syndrome in mouse models ([71]12–[72]15),
   and human retinal cells or cell lines ([73]16–[74]19). However, the
   pathogenic mechanisms that are responsible for Zika-associated anterior
   uveitis have not been investigated previously. In the anterior eye,
   iris pigment epithelial cells play a major role in controlling the
   immune microenvironment ([75]20). In herpes viral anterior uveitis,
   which is the most studied viral anterior uveitis, pathological changes
   involving these cells result in a variety of gross ocular changes,
   including tissue defects that may impact iris appearance ([76]21). To
   address the important knowledge gap around the pathogenesis of
   Zika-associated anterior uveitis, we investigated interactions between
   ZIKV and primary human iris pigment epithelial cells using infectivity
   assays and next generation sequencing of cellular RNA content
   (RNA-Seq).

Methods

Human Iris Pigment Epithelial Cells

   Iris pigment epithelial cell isolates were prepared from paired human
   cadaver donor eyes, using methods that were adapted from the technique
   published by Mai et al. ([77]22). Irises were carefully dissected from
   the two posterior eyecups, digested in 0.25% trypsin (Thermo Fisher
   Scientific-Gibco, Grand Island, NY) for 45 minutes at 37°C, and
   transferred to phosphate buffered saline (PBS) with 2% fetal bovine
   serum (FBS; Bovogen Biologicals, Keilor East, Australia) at room
   temperature. Pigment epithelial cells were gently brushed from the
   tissue using the bent tip of a Pasteur pipette. The cell suspension was
   pelleted at 10 x g for 5 minutes at room temperature, washed with PBS
   with 2% FBS, and re-suspended in Epithelial Cell Medium (ScienCell
   Research Laboratories, Carlsbad, CA; catalog number 4101, consisting of
   basal medium, 2% FBS, Epithelial Cell Growth Supplement and a
   penicillin-streptomycin solution). Cells were expanded at 37°C and 5%
   CO[2] in air, with medium refreshed twice a week, initially in a 6 cm
   diameter dish, subsequently trypsin-passaged and transferred to a T75
   flask, and finally stored frozen in liquid nitrogen on reaching
   confluence. Cell phenotype was confirmed for all cell isolates by
   immunocytochemical detection of cytokeratin-8 indicating epithelial
   lineage and absence of α-smooth muscle actin, which is expressed during
   mesenchymal differentiation (see Cytoimmunofluorescence). All cell
   isolates were demonstrated to be free of Mycoplasma species
   contamination by quantitative real-time polymerase chain reaction
   (qPCR) of DNA extracted from culture supernatant.

Zika Virus

   Zika virus (strain PRVABC59: National Center for Biotechnology
   Information Nucleotide (GenBank) Accession Number, [78]KU501215;
   originally isolated from human serum in Puerto Rico in 2015) was
   propagated in C6/36 mosquito cells (American Type Culture Collection
   [ATCC], Gaithersburg, MA) that were grown in Eagle’s basal medium, 1 mM
   sodium pyruvate, 1x MEM Non-Essential Amino Acids Solution, 2 mM
   L-glutamine and 10% FBS (all from Thermo Fisher Scientific-Gibco), and
   transferred to a medium modified by the removal of the amino acid
   supplement and reduction of FBS to 7.5%, and with the addition of 10 mM
   HEPES, ahead of infection. Virus stocks were titered by plaque assay on
   Vero cells, with plaques detected by neutral red overlay and expressed
   as plaque forming units/mL, as previously published ([79]23).

Viral Infection of Human Iris Pigment Epithelial Cells

   Human iris pigment epithelial cells were rapidly thawed and plated in
   10 cm diameter tissue culture dishes in Epithelial Cell Medium with 2%
   FBS. After cells reached confluence, they were subcultured to 6 cm
   dishes (2 x 10^6 cells/dish) or 6-well plates, including wells with
   coverslips (2 x 10^5 cells/well), and rested overnight. Subconfluent
   monolayers of iris epithelial cells were infected with ZIKV at
   multiplicity of infection (MOI) of 1 or 5, or mock-infected, in a
   minimum amount of FBS-free Dulbecco’s Modified Eagle Medium (DMEM,
   Thermo Fisher Scientific-Gibco; catalog number 11965). After 90
   minutes, the inoculum was removed, and the infected cultures were
   returned to incubation in fresh Epithelial Cell Medium for a total of
   24 hours, when cells were either lyzed with TRIzol Reagent (Thermo
   Fisher Scientific-Invitrogen, Carlsbad, CA) and stored at -80°C ahead
   of RNA extraction, or fixed with 2% paraformaldehyde for 30 minutes and
   stored in 70% ethanol at 4°C for cytoimmunofluorescence. Culture
   supernatant was snap-frozen at -80°C. A 24-hour time interval was
   selected to obtain a comprehensive picture of the host cell response to
   replicating virus. All work with live virus was conducted in a Physical
   Containment Level 2 Facility, under a Microbiological Dealing issued by
   the Flinders University Institutional Biosafety Committee (dealing
   number: 2016-07.1).

Focus Forming Assay

   Supernatants from ZIKV-infected and uninfected human iris pigment
   epithelial cell monolayers were tested for infectious virus with a high
   throughput plaque assay performed on an Operetta High-Content Analysis
   System (PerkinElmer, Waltham, MA), modifying a previously reported
   method ([80]24). In brief, Vero cells were seeded at 1 x 10^4 cell/well
   in 96-well plates and rested overnight, subsequently exposed in
   triplicate to serially diluted supernatant –or medium only control or
   ZIKV (3 x 10^6 pfu/mL) positive control– for 90 minutes, and finally
   incubated in fresh medium for 24 hours. Cell monolayers were fixed with
   2% paraformaldehyde, permeabilized with 0.05% IGEPAL CA-630, and
   labeled with mouse anti-flavivirus envelope protein (ENV) antibody
   (ATCC HB-112TM hybridoma [clone 4G2] culture supernatant diluted 1:10,
   applied overnight at 4 °C), followed by Alexa Fluor 488-tagged donkey
   anti-mouse immunoglobulin antibody (Thermo Fisher Scientific-Molecular
   Probes, Eugene, OR; catalog number A21202; working concentration, 10
   µg/mL, applied for 1 hour at room temperature) with Hoechst 33342
   (2’-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5’-bi-1H-benzimidazol
   e trihydrochloride trihydrate) counterstain. Wells were imaged using
   the Operetta High-Content Analysis System (11 fields/well, 100x
   magnification), and images were analyzed using the Columbus Image Data
   Storage and Analysis v2.7 (PerkinElmer) to quantify the number of
   labeled infected cell clusters per well. Data was processed in Vortex
   v2014.03.71496.59-s (Dotmatics, Bishops Stortford, UK), including
   calculation of focus forming units/mL for each supernatant sample and
   dilution.

Cytoimmunofluorescence

   Human iris pigment epithelial cell monolayers were fixed with 2% or 4%
   paraformaldehyde in preparation for cytoimmunofluorescence labeling.
   For cell phenotyping, monolayers were labeled overnight at 4°C with one
   of following rabbit polyclonal antibodies diluted in 0.05% Triton X-100
   and 2% bovine serum albumin in PBS: anti-human cytokeratin 8 (Abcam,
   Cambridge, United Kingdom; catalog number ab53280; working dilution,
   1:250, equivalent to 0.132 µg/mL), α-smooth muscle actin (Abcam,
   catalog number ab5694; working dilution, 1:100, equivalent to 2 µg/mL)
   and rabbit immunoglobulin (Vector Laboratories, Burlingame, CA; catalog
   number I-1000, working concentration, 2 µg/mL). Cell monolayers were
   incubated with Alexa Fluor 488-tagged donkey anti-rabbit immunoglobulin
   antibody (Thermo Fisher Scientific-Molecular Probes; catalog number
   A11008; working concentration, 1 µg/mL) for 1 hour at room temperature,
   counterstained with DAPI (4′,6-diamidino-2-phenylindole) for 2 minutes,
   and imaged by fluorescence microscopy at 200x magnification. For
   demonstration of viral infection, ZIKV-infected and uninfected cell
   monolayers were labeled for 1 hour at room temperature with mouse
   anti-flavivirus envelope protein antibody, used as hybridoma culture
   supernatant diluted 1:10 in Hanks’ balanced salt solution with 2% goat
   serum. Cell monolayers were incubated with Alexa Fluor 488-tagged goat
   anti-mouse immunoglobulin antibody (Thermo Fisher Scientific-Molecular
   Probes; catalog number A11001; working concentration, 13 µg/mL) for 30
   minutes at room temperature, counterstained with Hoechst 33342 for 10
   minutes, and imaged by confocal microscopy at 100x magnification.

RNA Extraction

   Total RNA was recovered from ZIKV-infected and uninfected iris pigment
   epithelial cells by phenol/chloroform extraction using Trizol Reagent,
   according to the manufacturer’s protocol. RNase-free glycogen was added
   to all samples during precipitation to maximize RNA yields. RNA was
   frozen at -80°C prior to use for RNA-Seq and reverse transcription
   (RT)-qPCR. Nucleic acid concentrations were determined using the Qubit
   2.0 Fluorometer (Thermo Fisher Scientific-Life Technologies, Carlsbad,
   CA).

Reverse Transcription-Polymerase Chain Reaction

   Reverse transcription was carried out using iScript Reverse
   Transcription Supermix for RT-qPCR (Bio-Rad, Hercules, CA). Total RNA
   input was 500 ng per reaction, yielding 20 μl of cDNA which was diluted
   10-fold ahead of use in qPCR.

   Quantitative real-time PCR was performed using the CFX Connect Real
   Time PCR Detection System (Bio-Rad), and a reaction mix consisting of 4
   μL of iQ Sybr Green Supermix or SsoAdvanced SYBR Green Supermix
   (Bio-Rad), 1.5 μL each of 20 μM forward and reverse primer, 11 μL of
   nuclease-free water and 2 μL of diluted cDNA. Primer sequences (and
   expected product sizes) were as follows: ZIKV ENV forward
   5’-GCTGGDGCRGACACHGGRACT-3’, reverse 5’-RTCYACYGCCATYTGGRCTG-3’ (304
   bp); interferon-β (IFN-β) forward 5’-AAACTCATGAGCAGTCTGCA-3’, reverse
   5’-AGGAGATCTTCAGTTTCGGAGG-3’ (168 bp); peptidylprolyl isomerase A
   (PPIA) forward 5’-GAGCACTGGAGAGAAAGGATTT-3’, reverse
   5’-GGTGATCTTCTTGCTGGTCTT-3’ (355 bp); and ribosomal protein lateral
   stalk subunit P0 (RPLP0) forward 5’-GCAGCATCTACAACCCTGAA-3’, reverse
   5’-GCAGATGGATCAGCCAAGAA-3’ (235 bp). Primer efficiency for all primer
   sets was greater than 85.0% except in the case of ZIKV ENV, for which
   efficiency was 76.5%. Melting curves were included in each RT-qPCR run
   to confirm a single peak was produced, and amplicon sizes for all
   primers were confirmed by agarose gel electrophoresis.

   Absolute quantification of viral genome equivalents was carried out by
   generating a standard curve for ZIKV ENV using purified PCR product
   serially diluted 10-fold from an initial concentration of 10 pg/μL.
   Starting quantity in ng (SQ) was determined for ZIKV-infected and
   uninfected samples using CFX Manager software v3.1 (Bio-Rad). Viral
   genome equivalent number was calculated using the equation
   ([SQ/10^9]/[bp molecular mass x product size]) x 6.02214 x 10^23.
   Relative expression of IFN-β was determined by the Pfaffl method
   ([81]25) and normalized to two reference genes –PPIA and RPLP0– which
   were stable as indicated by CV-value less than 0.25 and M-value less
   than 0.5.

RNA Sequencing

   An RNA integrity number of at least 8 was confirmed on the LabChip GX
   Touch 24 Nucleic Acid Analyzer (Perkin Elmer, Hopkinton, MA). The cDNA
   libraries were prepared with the TruSeq Stranded Total RNA Kit
   (Illumina, San Diego, CA), strictly in accordance with the
   manufacturer’s instructions. Pooled libraries were sequenced on the
   NextSeq 500, with the High-Output Kit for 2 x 75 cycles of sequencing,
   and aiming for an average depth of 50 x 10^6 reads per sample. The PhiX
   Control v3 library (Illumina) was used as a sequencing control.

Processing and Analysis of RNA Sequencing Data

   Quality of the data obtained by RNA sequencing was assessed with FastQC
   v0.11.3 ([82]26). Sequences were trimmed with Trimmomatic v0.38
   ([83]27) for minimum and maximum lengths of 50 and 75 nucleotides,
   respectively, and a 4-nucleotide sliding window with an average Phred
   score of 25. Reads passing quality control were aligned to the GRCh38
   human genome using HiSAT2 v2.1.0 ([84]28) set to default parameters.
   FeatureCounts v1.5.0-p2 ([85]29) was employed to count reads aligning
   to genes, as annotated in Ensembl v93, and counts of those genes
   uniquely mapped to the human genome were taken forward in the analysis.
   To investigate samples for contamination with non-human organisms,
   sequence reads were classified against the NCBI RefSeq database of
   bacterial, archaeal, viral and eukaryotic microbial genomes, as well as
   the human genome, using Kraken2 ([86]30).

   Further statistical analyses and visualization were performed in R
   v3.5.0 ([87]31). The EdgeR v3.22.3 ([88]32) package from Bioconductor
   was used to perform library size normalization (with the trimmed mean
   of M-values [TMM] method), multi-dimensional scaling, and differential
   expression analyses (with the glmQFit function). Variation in the data
   from unknown sources was examined using svaseq ([89]33), and
   differential expression analysis was performed in a genewise negative
   binomial generalized linear model in EdgeR that controlled for donor
   effect (see Data Availability Statement for link to BitBucket data
   repository). Differentially expressed genes were defined by two-fold or
   greater change, and a Benjamini and Hochberg corrected p-value or false
   discovery rate (FDR) ([90]34) less than 0.05. Pathway and Gene Ontology
   enrichment analyses were performed in InnateDB ([91]35). To identify
   differentially expressed genes associated with estimated viral load,
   host gene expression was correlated with read counts per million
   assigned to the ZIKV genome in each sample by Kraken2 (Spearman
   correlation, p-value less than 0.05). Transcription factor binding site
   analysis was performed using the findMotifs.pl program in HOMER v4.10
   ([92]36), with the human promoter set. A network of molecular
   interactions between differentially expressed genes, products encoded
   by those genes and/or their first neighbor interactors was constructed
   using the Contextual Hub Analysis Tool (CHAT) ([93]37) in Cytoscape
   v3.8.1 ([94]38), with annotations by Interferome v2.0 ([95]39) and
   InnateDB ([96]35). This tool identifies “nodes” in the network that are
   more highly connected to differentially expressed genes than is
   expected by chance alone (FDR less than 0.05).

Results

   Iris pigment epithelial cells were sourced from 12 men and 8 women
   cadaveric donors, whose ages at death ranged from 44 to 78 years
   (median = 61.5 years). Time from death to processing of the human
   eyecups ranged from 7 to 23 hours (median = 12.5 hours), and iris
   pigment epithelial cells were isolated over a period of 13 to 19 days
   (median = 16 days). The cells presented as rounded bodies, with some
   spindle morphology when plated subconfluent ahead of infection ([97]
   Figure 1A ). Across all 20 isolates, cytoimmunofluorescence on cells
   from the same passage as used for subsequent infections indicated
   homogeneous expression of cytokeratin-8 and complete absence of
   α-smooth muscle actin, indicating highly pure epithelial cell isolates
   with no evidence of mesenchymal differentiation ([98] Figure 1B ).

Figure 1.

   [99]Figure 1
   [100]Open in a new tab

   (A) Light photomicrograph of human iris pigment epithelial cells
   immediately prior to infection. Original magnifications: 40x (main
   image) and 100x (inset image); Scale bars: 500 µm (main image) and 200
   µm (inset image). (B) Fluorescence photomicrographs of human iris
   pigment epithelial cells immunolabeled to detect presence of
   cytokeratin 8 (CK8) and absence of alpha-smooth muscle actin (SMA),
   with negative control labeled with species-matched immunoglobulin
   (IgG). Alexa Fluor 488 (green) with DAPI nuclear counterstain (blue).
   Original magnification: 200x; Scale bar: 100 µm. (C) Fluorescence
   photomicrographs of ZIKV-infected and uninfected human iris pigment
   epithelial cells immunolabeled to detect flavivirus envelope protein.
   Alexa Fluor 488 (green) with Hoechst 33342 nuclear counterstain (blue).
   Original magnification: 100x; Scale bar: 100 µm. (D) Correlation plots
   showing cellular viral load in genome equivalents and infectious foci
   generated from culture supernatant for human iris pigment epithelial
   cells infected for 24 hours with ZIKV (multiplicity of infection = 1
   and 5). Circles represent individual cell isolates from 20 donors. Line
   indicates trend. Data were analyzed by Pearson correlation coefficient.
   (E) Graph presenting normalized expression of interferon (IFN)-β
   transcript, calculated relative to peptidylprolyl isomerase A (PPIA)
   and ribosomal protein lateral stalk subunit P0 (RPLP0), in human iris
   pigment epithelial cells infected for 24 hours with ZIKV (multiplicity
   of infection = 5). Circles represent individual cell isolates from 20
   donors. Crossbars indicate mean, and error bars indicate standard
   deviation. Data were analyzed by Wilcoxon signed-rank test.

   To investigate the susceptibility of human iris pigment epithelial
   cells to infection with ZIKV, subconfluent cell monolayers were exposed
   to the PRVABC59 strain for 24 hours at an MOI of 1.
   Cytoimmunofluorescent labeling of cell monolayers for the flavivirus
   ENV identified ZIKV in all 20 isolates following exposure, with viral
   antigen detected in between 4.4% and 43.2% (median = 14.3%) of cells
   across the isolates ([101] Figure 1C ). The RT-qPCR analysis of
   cellular RNA and focus forming assay of culture supernatant also
   indicated productive infection of cells at 24 hours, with obvious
   variation in susceptibility between the 20 isolates, and this result
   was replicated at an MOI of 5 ([102] Figure 1D ). For an initial
   assessment of the type I IFN response of human iris pigment epithelial
   cell to infection with ZIKV, IFN-β was measured by RT-qPCR in isolates
   infected at an MOI of 5. This indicated substantial, but variable,
   increase in cellular IFN-β transcript following infection ([103] Figure
   1E ). These results demonstrated that human iris pigment epithelial
   cells were moderately susceptible to ZIKV infection, and suggested that
   this cell population mounted a strong anti-viral response. The results
   also indicated considerable variation in these parameters across cells
   isolated from eyes of different individuals.

   To explore the molecular response of human iris pigment epithelial
   cells to infection with ZIKV at a whole transcriptome level, RNA-Seq
   was performed on total RNA extracted from the 20 primary cell isolates
   24 hours following infection with the PRVABC59 strain at an MOI of 5.
   Across the samples from uninfected isolates, there were 33.6 x 10^6
   mean paired reads, with 33.4 x 10^6 (99.5%) of reads aligned to the
   human genome, and 25.0 x 10^6 (74.5%) mean paired reads assigned to
   annotated genes. After correction for library size differences, reads
   aligned to the flavivirus genome in samples infected with ZIKV averaged
   9.9% (range = 2.0-24.9%) ([104] Supplementary Figure 1 ). Comparison
   with a RefSeq database of bacterial, archaeal, viral and eukaryotic
   genomes indicated negligible contamination with other organisms,
   including Mycoplasma species. Alignment statistics are presented in
   [105]Supplementary Table 1 .

   Multidimensional scaling of the gene expression data indicated clear
   separation of the total RNA transcriptome of ZIKV-infected versus
   non-infected human iris pigment epithelial cell isolates ([106] Figure
   2A ). Data visualization with svaseq ([107]33) indicated that the
   largest source of variation in the data related to donor (Kruskal
   Willis, p = 0.005), but this was not explained on the basis of donor
   age at death or sex, or time from death to cell isolation, and also was
   not related to batch. Thus, in order to focus on the common cellular
   response to ZIKV, differential expression analysis was performed
   controlling for the impact of donor. A total of 7,935 genes (47% of all
   annotated genes) were differentially expressed between infected and
   uninfected cells at FDR less than 0.05, including 606 genes that were
   increased and 7 genes that were decreased at least two-fold in the
   infected cells ([108] Figure 2B , [109]Supplementary Table 2 ). Heat
   maps showed obvious separation of the set of all differentially
   expressed genes between ZIKV-infected and uninfected samples ([110]
   Figure 3A ), although the 1,000 most variable genes were less
   discriminating ([111] Figure 3B ).

Figure 2.

   Figure 2
   [112]Open in a new tab

   (A) Multidimensional scaling plots showing global gene expression in
   ZIKV-infected (red) and uninfected (blue) human iris pigment epithelial
   cell isolates. Numbers indicate the 20 individual donors from whose
   eyes each isolate was generated. PC = principal component. (B) Volcano
   plot displaying differentially expressed genes between ZIKV-infected
   and uninfected human iris pigment epithelial cells, based on normalized
   counts per million (false discovery rate < 0.05 and fold change > 2).
   Red dots indicate significantly upregulated transcripts, and blue dots
   indicate significantly downregulated transcripts.

Figure 3.

   Figure 3
   [113]Open in a new tab

   (A) and (B) Heat maps indicating normalized counts per million from
   sequencing of total RNA from ZIKV-infected (red) and uninfected (blue)
   human iris pigment epithelial cells isolated from eyes of 20 individual
   donors. Z-score is shown for (A) all differentially expressed genes and
   (B) the 1,000 most variable genes. Color scale changes from orange to
   blue, with higher intensity orange representing higher counts for each
   transcript. (C–E) Graphs presenting the 15 most enriched categories –by
   (C) Reactome or KEGG pathways, (D) Gene Ontology biological processes
   and (E) Gene Ontology molecular functions– for genes that were
   upregulated in ZIKV-infected cells, as annotated in InnateDB ([114]35).

   Pathway and Gene Ontology biological process analyses of genes
   significantly upregulated (ie. two-fold or greater change and FDR less
   than 0.05) in ZIKV-infected iris pigment epithelial cells compared to
   uninfected cells revealed a strong transcriptional response to
   infection, involving over 200 different pathways or processes.
   Molecules involved in immune functions were highly enriched among
   differentially expressed genes. More specifically, differentially
   expressed genes were enriched for groupings that included activation of
   pathogen recognition systems –with retinoic acid-inducible gene I
   (RIG-I)-like receptor (RLR) and toll-like receptor (TLR) signaling– and
   anti-viral and inflammatory cytokine responses –with type I and type II
   IFN signaling ([115] Figures 3C, D , [116]Supplementary Table 3 ). Gene
   ontology molecular function analysis of differentially expressed genes
   similarly indicated an active response to the virus. Highly enriched
   functions included broad terms, such as cytokine and chemokine
   activities, as well as more specific categories, such as single- and
   double- stranded RNA binding –signifying viral recognition– and
   2′,5′-oligoadenylate synthetase (OAS) activity and tumor necrosis
   factor (TNF) receptor binding –consistent with anti-viral responses
   ([117] Figure 3E , [118]Supplementary Table 3 ). Transcription factor
   binding site analysis identified 13 regulatory motifs that were
   enriched in promoter regions of significantly upregulated genes,
   including multiple IFN-regulating factors ([119] Table 2 ). No binding
   sites were enriched in genes with significantly downregulated
   expression.

Table 2.

   Transcription factor binding sites that were enriched in promoters of
   highly up-regulated genes (target sequences) in comparison with all
   genes (background sequences) in human iris pigment epithelial cells 24
   hours following infection with ZIKV.
   Motif (Family) Consensus sequence Number of target sequences with motif
   (%) Number of background sequences with motif (%) False discovery rate
   ISRE (IRF) AGTTTCASTTTC 67 (9.6) 427.5 (1.0) <0.0001
   IRF1 (IRF) GAAAGTGAAAGT 85 (12.2) 864.1 (2.0) <0.0001
   IRF2 (IRF) GAAASYGAAASY 76 (10.9) 759.3 (1.8) <0.0001
   p65/RELA (RHD) GGAAATTCCC 29 (4.2) 404.1 (1.0) <0.0001
   IRF4 (IRF) ACTGAAACCA 71 (10.2) 2057.2 (4.8) <0.0001
   T1ISRE (IRF) ACTTTCGTTTCT 10 (1.4) 42.6 (0.1) <0.0001
   bZIP-IRF (bZIP, IRF) NAGTTTCABTHTGACTNW 61 (8.7) 1706.1 (4.0) <0.0001
   p65/RELA (RHD) WGGGGATTTCCC 95 (13.6) 3490.7 (8.2) <0.0001
   PU.1-IRF (ETS, IRF) MGGAAGTGAAAC 194 (27.8) 8745 (20.5) 0.0001
   PRDM1 (Zf) ACTTTCACTTTC 78 (11.2) 2838.8 (6.6) 0.0002
   Tbx20 (T-box) GGTGYTGACAGS 28 (4.0) 885 (2.1) 0.0234
   Meis1 (Homeobox) VGCTGWCAVB 167 (23.9) 8238.7 (19.3) 0.0331
   [120]Open in a new tab

   BZIP, basic leucine zipper domain; ETS, erythroblast
   transformation-specific; IRF, interferon regulatory factor; ISRE,
   interferon-stimulated response element; Meis1, Meis homeobox 1; NF-kB
   subunit; PRDM1, PR domain-containing protein 1; PU.1, PU box binding-1;
   RELA, RelA protooncogene, RHD, rel homology domain; T1ISRE, type I
   interferon-stimulated response element; Tbx20, T-box transcription
   factor 20; Zf, zinc finger.

   A system-level CHAT network ([121]37) was built around genes that were
   significantly differentially expressed between ZIKV-infected and
   uninfected human iris pigment epithelial cells, including
   first-neighbor interactors. This network included 6275 nodes ([122]
   Figure 4 , [123]Supplementary Table 4 ). There were 24 contextual hubs
   that interacted with differentially expressed genes more frequently
   than predicted by chance alone ([124] Table 3 ). The most highly
   connected contextual hubs –with greater than 15 contextual neighbors
   and greater than 200 total neighbors– were members of the Janus kinase
   (JAK)-signal transducer and activator of transcription (STAT), or
   nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)
   intracellular signaling pathways, indicating their involvement in type
   I IFN anti-viral responses and inflammation. These six hubs included:
   inhibitor of NF-κB kinase, regulatory subunit γ (IKBKG); rela
   protooncogene, NF-κB subunit (RELA); TNF receptor-associated factor 6
   (TRAF6); STAT1; STAT3; and ubiquitin-like modifier ISG15 (ISG15).
   Receptor-interacting serine/threonine kinase 1 (RIPK1) was the most
   significantly connected contextual hub (p = 5.49 x 10^-5), with 16
   contextual neighbors and 102 total neighbors.

Figure 4.

   [125]Figure 4
   [126]Open in a new tab

   Network of interactions between differentially expressed genes and the
   first-order interactors of these genes or their encoded products in
   ZIKV-infected human iris pigment epithelial cell isolates from 20
   individual donors. CHAT ([127]37) identified nodes in the network that
   were connected to differentially expressed genes more than expected by
   chance. Node size is scaled to -log[10] of the false discovery rate
   where larger nodes are those that were identified as being more
   statistically significant. Purple nodes are type I interferon
   (IFN)-stimulated genes (ISGs); green nodes are type II IFN ISGs; red
   nodes are type I and II IFN ISGs; blue nodes are not IFN-responsive, as
   annotated by Interferome v2.0 ([128]39). Diamond-shaped nodes represent
   differentially expressed genes (false discovery rate < 0.05 and
   fold-change > 2). Edges between nodes represent molecular interactions,
   as annotated by InnateDB ([129]35). Type I and II IFN ISGs were
   statistically over-represented in this network. Hypergeometric test, p
   < 0.0001.

Table 3.

   Contextual hubs (highly connected molecular nodes) that were found to
   interact with differentially expressed genes more frequently than
   expected by chance in human iris pigment epithelial cells 24 hours
   following infection with ZIKV.
   Contextual hub Contextual neighbors Total neighbors False discovery
   rate
   RIPK1 16 102 5.49E-05
   RNF31 13 83 4.68E-04
   MAVS 14 96 4.68E-04
   RBCK1 12 75 8.10E-04
   STAT3 24 297 0.0013
   RELA 25 329 0.0020
   BIRC3 12 87 0.0024
   HLA-C 11 75 0.0029
   DBP 5 11 0.0034
   SHARPIN 8 41 0.0052
   STAT1 18 207 0.0052
   ISG15 18 218 0.0093
   HLA-G 5 15 0.0129
   TLR3 7 35 0.0129
   UBE2L6 8 49 0.0151
   IKBKE 11 98 0.0196
   SRA1 7 39 0.0219
   IKBKG 25 417 0.0377
   BIRC2 13 146 0.0400
   CASP1 7 44 0.0415
   TNFAIP3 10 92 0.0428
   TIRAP 7 45 0.0436
   CXCR3 3 5 0.0446
   TRAF6 21 330 0.0446
   [130]Open in a new tab

   BIRC3, baculoviral IAP repeat-containing protein; CASP1, caspase 1,
   apoptosis-related cysteine protease; CXCR3, chemokine, CXC motif,
   receptor 3; DBP , d-box-binding PAR bZIP transcription factor; HLA ,
   major histocompatibility complex, class I; IKBK , inhibitor of nuclear
   factor kappa-B kinase; ISG15, ubiquitin-like modifier ISG15 (a.k.a.
   interferon-induced protein 15); MAVS, mitochondrial antiviral signaling
   protein; RBCK1, RANBP-type and C3HC4-type zinc finger-containing 1;
   RELA, rela protooncogene, NFKB subunit; RIPK1, receptor-interacting
   serine/threonine kinase 1; RNF31, ring finger protein 31; SHARPIN,
   shank-associated RH domain interactor; SRA1, steroid receptor RNA
   activator 1; STAT, signal transducer and activator of transcription;
   TIRAP, TIR domain-containing adaptor protein; TLR3, toll-like receptor
   3; TNFAIP3, tumor necrosis factor-alpha-induced protein 3; TRAF6, TNF
   receptor-associated factor 6; UBE2L6, ubiquitin-conjugating enzyme E2L
   6.

   Associations between gene expression by the host cell and intracellular
   ZIKV load were identified by correlating upregulated genes with
   normalized read counts assigned to the viral genome in the infected
   iris pigment epithelial cell isolates across the 20 human donors. This
   analysis identified 146 upregulated genes that were significantly
   correlated with read counts assigned to ZIKV, including 122 that were
   positively correlated and 24 that were negatively correlated ([131]
   Supplementary Table 5 ). Reactome pathway enrichment analysis of these
   genes indicated ‘IFN signaling’ and ‘immune system’ were negatively
   correlated with ZIKV read counts, while cell cycle-related pathways
   were positively correlated ([132] Figure 5A ). Examples of upregulated
   IFN-stimulated genes that were negatively correlated with ZIKV load
   included: adenosine deaminase, RNA-specific (ADAR); IFN-induced protein
   44 (ITI44); IFN-induced protein with tetratricopeptide repeats 5
   (IFIT5); and OAS1 ([133] Figure 5B ).

Figure 5.

   Figure 5
   [134]Open in a new tab

   (A) Correlations between genes upregulated in ZIKV-infected human iris
   pigment epithelial cells and read counts per million assigned to the
   ZIKV genome (ZIKV load). Spearman correlation, p < 0.05. Gray circles
   indicate upregulated genes. Red lines indicate positive correlations,
   and blue lines indicate negative correlations. Enriched Reactome
   pathways achieving false discovery rate < 0.05 are listed left of the
   relevant grouping, with annotations by InnateDB ([135]35). (B) Examples
   of negative correlations between interferon (IFN)-stimulated genes and
   ZIKV load. Red dots indicate 20 individual donors. Line indicates
   trend. ADAR, adenosine deaminase, RNA-specific; IFI44, IFN-induced
   protein 44; IFIT5, IFN-induced protein with tetratricopeptide repeats
   5; OAS1, 2’-5’-oligoadenylate synthetase 1.

Discussion

   Zika virus disease is often asymptomatic or manifest as a mild and
   self-limited systemic illness in adult patients. However, serious
   inflammatory complications may occur, and these include anterior
   uveitis, which has been linked with presence of ZIKV in the anterior
   eye ([136]8, [137]9). Our work represents the first research to examine
   mechanisms of Zika-associated anterior uveitis, focusing on the
   interactions between the virus and iris pigment epithelial cells, and
   using the PRVABC59 strain isolated during the American epidemic and
   primary cells isolated within 24 hours of death from human cadaveric
   donors. Human iris pigment epithelial cell isolates generated from 20
   donors were permissive to ZIKV infection, and supported replication and
   release of the virus. RNA-Seq indicated a vigorous molecular response
   of the host cell to ZIKV. Almost 50% of annotated genes were
   differentially expressed between infected and uninfected cells, and 99%
   of the 613 genes that changed at least two-fold were up-regulated.
   Pathway, Gene Ontology and network analyses indicated strong activation
   of viral recognition and defense, as well as biosynthesis mechanisms
   consistent with production of virus. Although all iris pigment
   epithelial cell isolates mounted a type I IFN response, intracellular
   virus load correlated inversely to overall IFN signaling across the
   donors.

   When uveitis occurs in the context of a systemic infectious disease,
   the pathology usually represents a combination of tissue damage induced
   by the microbe and reactive inflammation by the host. Although some
   forms of infection-associated uveitis are believed to be primarily
   immune-mediated ([138]40), recent widespread molecular testing of
   ocular fluids has demonstrated that a broad range of both dangerous and
   innocuous viruses access the eyes in immunocompetent persons to cause
   uveitis –for example, cytomegalovirus (CMV), rubella virus and Ebola
   virus (EBOV) ([139]41–[140]43). ZIKV also has been recovered from the
   aqueous humor in anterior uveitis ([141] Table 1 ). Studies in mice
   suggest that ZIKV accesses the eye from the blood stream in myeloid
   cells that become hypermotile ([142]44, [143]45), as is also the case
   for more common ocular pathogens such as Toxoplasma gondii ([144]46).
   Various ocular cell populations are preferred hosts for different
   organisms, including retinal pigment epithelium for EBOV and
   Mycobacterium tuberculosis ([145]47, [146]48), and retinal Müller glial
   cells for CMV and T. gondii ([147]49, [148]50). Given that ZIKV causes
   serious retinal pathology in utero, previous research in the field has
   focused on cells of the posterior eye, including Müller cells, retinal
   pigment epithelial cells and retinal vascular endothelial cells
   ([149]17, [150]19, [151]51). Iris pigment epithelial cells are key
   players in inflammation of the anterior segment of the eye, which
   defines anterior uveitis. They have a dual role, as key regulators of
   ocular immune privilege ([152]20), and as the source of an array of
   inflammatory cytokines ([153]52). Our data indicate that ZIKV is
   capable of productively infecting human iris pigment epithelial cells.

   While all 20 primary human iris pigment epithelial cell isolates were
   susceptible to infection with ZIKV, infectivity varied across donors,
   as demonstrated by RT-qPCR measurement of intracellular viral load and
   a focus forming assay of supernatant collected from cultures of
   infected cells. Variation in response to infection, as well as at
   baseline, is likely to be caused by multiple technical and biological
   factors. A consistent method of cell isolation and culture was used,
   but donor eyes were necessarily sourced on different dates. There was
   also unavoidable variation in time from death to cell isolation and
   time in culture, although these intervals were minimized to avoid
   mesenchymal transition of cells. Biological variation relates to
   individual genetics and epigenetics, as well as health and disease
   status. In work with other primary human ocular cells –including
   retinal and choroidal endothelial cells, Müller cells and retinal
   pigment epithelial cells– we ([154]53–[155]55) and other groups
   ([156]56–[157]59) have demonstrated variation in gene expression by
   human donor. This highlights the importance of not relying on results
   from individual cell isolates in drawing conclusions about
   pathobiological mechanisms. A major strength of our study is the large
   number of human donors, affording a comprehensive overview of the gene
   expression of the human iris pigment epithelium by RNA-Seq, while also
   identifying inter-individual variations.

   Intracellular viral recognition is coordinated by two pattern
   recognition receptor families: TLRs and RLRs ([158]60). Pathway and
   Gene Ontology analyses indicated high activation of RIG-I and melanoma
   differentiation-associated protein 5 (MDA5) signaling; TLR3, which
   recognizes double-stranded RNA specifically, was identified as a
   contextual hub by CHAT network analysis. Viral defense relies on IFN
   responses. IFN-β –the prototype type I IFN– was the most highly
   differentially expressed transcript in infected human iris pigment
   epithelial cells, with an approximately 900-fold upregulation across
   the 20 isolates. Enrichment analyses identified activation of type II
   IFN signaling, as well as type I. Type III IFNs –including IFN-λ1,
   IFN-λ2 and IFN-λ3– also were substantially increased following
   infection. The IFNλ receptor is a heterodimer of IL10RB, which is
   ubiquitously expressed, and IFNLR1, which has considerably restricted
   expression, but was identified in this cell population by RNA-Seq. The
   type III IFN system protects barriers ([159]61), including the female
   reproductive tract in Zika ([160]62). Recent work has identified Type
   III IFN signaling on the ocular surface, with corneal epithelium also
   expressing both subunits of IFNλR1 ([161]63). Expression of type III
   IFNs and IFNR1λ is consistent with the role of iris pigment epithelial
   cells in maintaining ocular immune privilege. The transcription factor
   analysis also indicated activity in pathways that would increase
   IFN-stimulated genes. Interestingly, key players in noncanonical NF-κB
   signaling were upregulated, suggesting the possibility of negatively
   regulating induction of type I IFNs ([162]64). NF-κB signaling also
   would be expected to promote inflammatory responses and trigger a
   breach in immune privilege, consistent with the development of anterior
   uveitis.

   While anti-viral IFN responses were highly upregulated in ZIKV-infected
   iris pigment epithelial cells, the intracellular viral load correlated
   inversely with IFN signaling. Consistently, multiple ISGs were
   upregulated at a relatively low level in heavily infected isolates.
   This result suggests ZIKV directly interferes with IFN signaling in
   epithelial cells. The ZIKV polypeptide yields 7 nonstructural (NS)
   proteins in addition to 3 structural proteins ([163]65). ZIKV NS5 in
   particular has multiple actions that target IFN signaling. On binding
   to RIG-I, NS5 represses the ubiquitination that promotes RIG-I-mediated
   signaling ([164]66). NS5 binds TANK-binding kinase 1 (TBK1),
   interfering with its ability to phosphorylate and activate IRF3 ahead
   of nuclear translocation ([165]67); NS5 also binds IRF3 to limit its
   activity ([166]68). Together with IRF9, STAT1 and STAT2 form the
   IFN-stimulated gene actor-3 complex (ISGF-3), but NS5 targets STAT2 for
   proteosomal degradation, by either binding it directly or binding the
   E3 ubiquitin ligase, seven in absentia homolog 2 (SIAH2), which
   subsequently binds STAT2 ([167]69, [168]70). Other nonstructural
   proteins also may impact IFN production and/or induction of ISGs
   ([169]68, [170]71). While NS5 suppresses type I and III IFN signaling,
   it enhances type II IFN signaling by destabilizing STAT2 and limiting
   formation of ISGF-3, and promoting formation of STAT1-STAT1 homodimers
   that bind γ-activated sequence (GAS) elements ([171]72). Thus,
   increased viral replication could promote anterior uveitis by
   increasing type II IFN signaling. NS5 also facilitates the assembly of
   the NLRP3 (NLR family, pyrin domain-containing 3) inflammasome
   ([172]73).

   The “central controller” of cell fate –RIPK1 ([173]74)– was the most
   significant contextual hub in the molecular network generated from the
   list of 613 differentially expressed genes in iris pigment epithelial
   cells following ZIKV infection. While not differentially expressed per
   our definition of two-fold change and FDR less than 0.05 ([174]
   Supplementary Table 2 ), RIPK1 increased highly significantly (FDR = 8
   x 10^-30) by 1.5-fold post-infection. Originally described as an
   activator of necroptosis, involvement of RIPK1 in different
   pathological processes is now known to be multifaceted. The protein has
   a C-terminal death domain, an N-terminal serine/threonine kinase
   domain, and an intermediate domain that participates in NF-κB
   activation ([175]74). In addition to necroptosis, RIPK1 may trigger
   apoptosis and promote inflammation, and conversely, it may act as a
   regulator of each of these processes ([176]75–[177]77). The ultimate
   impact of RIPK1 signaling in ZIKV-infected human iris pigment
   epithelial cells is thus difficult to predict. Recent work in a mouse
   central nervous system model of ZIKV infection indicated RIPK1
   signaling directed a “metabolic reprogramming” in infected neurons
   characterized by inhibition of succinate dehydrogenase, which
   suppressed viral replication ([178]78), suggesting RIPK1-controlled
   activities might have anti-viral effects.

   To interrogate the mechanisms of Zika-associated anterior uveitis as it
   occurs in the patient, we selected an in vitro system with iris pigment
   epithelial cells isolated from human cadaver eyes. Iris biopsy is not
   performed in the clinical setting, and thus it is not feasible to
   conduct such research using tissue from patients diagnosed with this
   condition. Removing the cells from the local microenvironment and the
   culture procedure is expected to induce some change in cell phenotype.
   We used early passage cell isolates, all of which were checked for
   presence of epithelial and absence of mesenchymal markers. The most
   common in vivo models are induced by systemic inoculation of virus into
   mice, including animals with deficient type I IFN signaling
   ([179]12–[180]15). A non-human primate model in the Rhesus macaque has
   recently been described ([181]79). However, these studies have not
   involved adult animals. Indeed, in descriptions of several mouse
   models, Zhao and colleagues ([182]13) commented that wild-type mice
   infected intraperitoneally with ZIKV lost susceptibility to ocular
   pathology from one week of age, and A129 mice –with null mutations in
   the IFN-α/β receptor– showed no ocular disease after 10 weeks. Past
   works also have focused largely on posterior eye pathology. An
   interesting new experimental model for investigating anterior uveitis
   involves injecting ZIKV into the aqueous humor of 1-month-old mice; the
   animals develop raised intraocular pressure, and pattern recognition
   receptors and inflammatory chemokines are induced in anterior segment
   tissues ([183]80).

   Zika-associated anterior uveitis occurs when ZIKV accesses the eye
   during a systemic infection. However, the eye is able to clear the
   virus, and the inflammation resolves either spontaneously or with the
   short-term use of corticosteroid eye drops. Our work represents the
   first observations around basic mechanisms of this emerging form of
   uveitis in a human-based experimental system. Our results suggest that
   the iris pigment epithelium is susceptible to ZIKV infection, but also
   mounts a rigorous pan-IFN response, which when combined with other
   elements of ocular immune privilege, may limit ocular pathology in the
   majority of affected individuals. Those persons in whom the virus
   replicates robustly within the iris pigment epithelium, may generate a
   stronger inflammatory response and thus be at higher risk of developing
   anterior uveitis.

Data Availability Statement

   The datasets presented in this study can be found in online
   repositories. Sequence data are available in the Gene Expression
   Omnibus (GEO) of the National Centre for Biotechnology Information
   (NCBI) under the accession number [184]GSE131605. Read counts and code
   are available in a BitBucket data repository:
   [185]https://bitbucket.org/lynnlab/ZIKV.

Ethics Statement

   Use of human cadaver donor eyes from the Eye Bank of South Australia
   (Adelaide, Australia) for this work was approved by the Southern
   Adelaide Clinical Human Research Ethics Committee (protocol number:
   175.13). Retrospective collection of the clinical data of individuals
   diagnosed with ZIKV-associated anterior uveitis at the Ophthalmology
   Clinic of Ribeirao Preto General Hospital, Ribeirao Preto, Sao Paulo,
   Brazil, between January 2016 and June 2016, was approved by the
   Hospital Ethics Committee of Human Research (protocol number:
   85195318.0.0000.5540).

Author Contributions

   JS conceived the study. FR, JC, JF, MM, BA, DL, and JS designed the
   study. JF and MS collected and synthesized the clinical data. JC, YM,
   LA, GO, GG, AD, BA and JS performed the laboratory experiments and/or
   analyzed laboratory data (including contributions to methodology). FR
   and DL analyzed and interpreted the RNA-Seq data. JS wrote the article
   including review of the relevant literature. FR and LA contributed
   substantially to preparation of the figures and/or tables in the
   article. FR, JC, JF, YM, LA, MS, GO, GG, AD, MM, BA and DL critically
   reviewed the content of the article. All authors contributed to the
   article and approved the submitted version.

Funding

   This work was supported by grants from Foundation for Support of
   Teaching, Research & Assistance of the Clinical Hospital, Faculty of
   Medicine of Ribeirão Preto - University of São Paulo (FAEPA: 1973/2019
   to JF); Avant Mutual Group (Doctor in Training Research Scholarship to
   GO); EMBL Australia (Group Leader Award to DL); Rebecca L. Cooper
   Medical Research Foundation (Research Grant to JS); National Health &
   Medical Research Council (GNT1139857 to JS); and Australian Research
   Council (FT130101648 to JS).

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Acknowledgments