Conjunctival epithelial cells resist productive SARS-CoV-2 infection.

Conjunctival epithelial cells, which express viral-entry receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine type 2 (TMPRSS2), constitute the largest exposed epithelium of the ocular surface tissue and may represent a relevant viral-entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of basal, suprabasal, and superficial epithelial cells, and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA sequencing (RNA-seq), with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-κB activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplantation.

In the eye of the storm: SARS-CoV-2 infection and replication at the ocular surface?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first emerged in December 2019 and spread quickly causing the coronavirus disease 2019 (COVID-19) pandemic. Recent single cell RNA-Seq analyses have shown the presence of SARS-CoV-2 entry factors in the human corneal, limbal, and conjunctival superficial epithelium, leading to suggestions that the human ocular surface may serve as an additional entry gateway and infection hub for SARS-CoV-2. In this article, we review the ocular clinical presentations of COVID-19 and the features of the ocular surface that may underline the overall low ocular SARS-CoV-2 infection. We critically evaluate the studies performed in nonhuman primates, ex vivo organ culture ocular models, stem cell derived eye organoids and the differences in infection efficiency observed in different parts of human ocular surface epithelium. Finally, we highlight the additional work that needs to be carried out to understand the immune response of the ocular surface to SARS-CoV-2 infection, which can be translated into prophylactic treatments that may be applied to other organ systems.

Co-expression of SARS-CoV-2 entry genes in the superficial adult human conjunctival, limbal and corneal epithelium suggests an additional route of entry via the ocular surface.

PURPOSE: The high infection rate of SARS-CoV-2 necessitates the need for multiple studies identifying the molecular mechanisms that facilitate the viral entry and propagation. Currently the potential extra-respiratory transmission routes of SARS-CoV-2 remain unclear. METHODS: Using single-cell RNA Seq and ATAC-Seq datasets and immunohistochemical analysis, we investigated SARS-CoV-2 tropism in the embryonic, fetal and adult human ocular surface. RESULTS: The co-expression of ACE2 receptor and entry protease TMPRSS2 was detected in the human adult conjunctival, limbal and corneal epithelium, but not in the embryonic and fetal ocular surface up to 21 post conception weeks. These expression patterns were corroborated by the single cell ATAC-Seq data, which revealed a permissive chromatin in ACE2 and TMPRSS2 loci in the adult conjunctival, limbal and corneal epithelium. Co-expression of ACE2 and TMPRSS2 was strongly detected in the superficial limbal, corneal and conjunctival epithelium, implicating these as target entry cells for SARS-CoV-2 in the ocular surface. Strikingly, we also identified the key pro-inflammatory signals TNF, NFKß and IFNG as upstream regulators of the transcriptional profile of ACE2+TMPRSS2+ cells in the superficial conjunctival epithelium, suggesting that SARS-CoV-2 may utilise inflammatory driven upregulation of ACE2 and TMPRSS2 expression to enhance infection in ocular surface. CONCLUSIONS: Together our data indicate that the human ocular surface epithelium provides an additional entry portal for SARS-CoV-2, which may exploit inflammatory driven upregulation of ACE2 and TMPRSS2 entry factors to enhance infection.

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes.

We investigated SARS-CoV-2 potential tropism by surveying expression of viral entry-associated genes in single-cell RNA-sequencing data from multiple tissues from healthy human donors. We co-detected these transcripts in specific respiratory, corneal and intestinal epithelial cells, potentially explaining the high efficiency of SARS-CoV-2 transmission. These genes are co-expressed in nasal epithelial cells with genes involved in innate immunity, highlighting the cells' potential role in initial viral infection, spread and clearance. The study offers a useful resource for further lines of inquiry with valuable clinical samples from COVID-19 patients and we provide our data in a comprehensive, open and user-friendly fashion at www.covid19cellatlas.org.