Zika Virus Infection of Human Iris Pigment Epithelial Cells.

Feargal J Ryan,Jillian M Carr,Milena Simões,Genevieve F Oliver,G Bracho Granado,João M Furtado,Binoy Appukuttan,David J Lynn,Michael Z Michael,Abby C Dawson,Justine R Smith,Yuefang Ma,Liam M Ashander

doi:10.3389/fimmu.2021.644153

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.

Highlights

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 [1]
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 multiplicity of infection (MOI) of 1
For an initial assessment of the type I IFN response of human iris pigment epithelial cell to infection with ZIKV, IFN-b was measured by reverse transcription (RT)-quantitative real-time polymerase chain reaction (qPCR) in isolates infected at an MOI of 5

Summary

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 [1]. 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 [2, 3], referred to as congenital Zika syndrome [4]. Characteristic abnormalities in this disease include severe microcephaly, thinned cerebral cortex with subcortical calcification, extrapyramidal motor disturbances and contractures. In some adults, infection with ZIKV results in serious inflammatory conditions [1]

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