SARS-CoV-2 infects human corneal epithelium and elicits an antiviral immune response

Purpose : We recently reported the presence of SARS-CoV-2 RNA, Spike, and Envelope proteins in the corneas of COVID-19 donors. However, the presence of viral RNA and antigens does not necessarily equate to infection. In this study, using RNA-seq of COVID-19 corneal tissue, and SARS-CoV-2 infection of human corneal epithelial cultures, we now examined whether SARS-CoV-2 could replicate in the cornea and elicits an innate immune response. Methods : Eyes from healthy and COVID-19 donors were from the Eversight eye bank. The corneal tissue was used for IHC detection of SARS-CoV-2 by RNA-FISH. In another experiment, total RNA was extracted from corneas for RNA-seq analysis to identify genes/ pathways altered by infection. In vitro studies were performed by infecting primary human corneal epithelial cells (HCECs) from normal and diabetic donor corneas with SARS-CoV-2. Bioinformatics analysis was performed to determine the differential gene expression. qPCR was used to assess the expression of innate inflammatory and antiviral genes and to confirm RNA-seq data in corneal tissue and cells. Results : RNA-FISH analysis showed the presence of both positive and negative strands of SARS-CoV-2 viral RNA in the epithelium of COVID-19 donor corneas. This coincided with infiltration of CD45+ cells in the stroma and induced expression of inflammatory (IL-6, IL1β) and antiviral (ISG15, OAS2) genes. RNA-seq analysis revealed significant upregulation of genes involved in the viral response, inflammation, and injury along with induction of lncRNA XIST and TSIX involved in modulation of the immune response. The primary HCECs were found permissive to SARS-CoV-2 infection, as evidenced by increase viral replication which peaked at day 3 p.i. along with an induction of p-STAT1. Interestingly, HCECs from diabetic cornea had higher viral RNA on day 1 p.i. compared to non-diabetic cells. SARSCoV-2 infected HCECs also exhibited induced expression of antiviral innate response genes, which was elevated in diabetic donor cornea cells. Conclusions : Our study confirms the presence of replicating SARS-CoV-2 viral RNA and antigen in the cornea of COVID-19 affected donors resulting in the production of inflammatory mediators and recruitment of CD45+ immune cells to the cornea. Moreover, HCECs from diabetic corneas had increased SARS-CoV-2 replication and immune response, suggesting that diabetes is a potential risk for ocular transmission of COVID-19.