Tracing the SARS-CoV-2 infection on the ocular surface: Overview and preliminary corneoscleral transcriptome sequencing

Abstract

COVID-19's impact on the ocular surface has already been recognized, however the molecular mechanisms induced by the infection on the ocular surface are still unclear. The aim of this paper is to provide a first overview of the transcriptional perturbations caused by SARS-CoV-2 on the ocular surface by analyzing gene expression profile of corneoscleral ring samples from post-mortem SARS-CoV-2 positive donors (PD). The presence of SARS-CoV-2 on the ocular surface, in tears and corneal tissues has rarely been detected in infected individuals in both the presence and the absence of ocular manifestations. In this preliminary study, 6 human corneoscleral tissues of 3 PD and two tissues from a negative donor (CTRL) were obtained at the local eye bank. The presence of genomic and sub-genomic SARS-CoV-2 RNAs was assessed by qRT-PCR, while transcriptome analysis (RNA-sequencing) was performed by Illumina. Principal Component Analysis (PCA), search for differentially expressed genes (DEGs) and Gene Ontology (GO)-enrichment analysis were performed. Three samples from PD were found positive for SARS-CoV-2 genomic RNA, although the absence of sub-genomic RNAs indicated an inactive virus. PCA analysis grouped 3 different clusters, one including CTRL, and the other two including, respectively, PD with undetected SARS-CoV-2 (PD-SARS-neg) and PD with detected SARS-CoV-2 (PD-SARS-pos). The DEGs in common with the 2 PD clusters included several genes associable to the interferon pathway, such as ADAMTS4, RSAD2, MMP1, IL6, ISG15 and proinflammatory cytokines. Among the down-regulated genes we found AQP5. GO analysis revealed 77 GO terms over-represented in PD-SARS-neg vs. CTRL, and 17 GO terms in PD-SARS-pos vs. CTRL. The presence of SARS-CoV-2 RNA and RNA-sequencing reads in ocular surface tissues supports the possibility that the eye acts as an entry route. The modulation of early responsive genes, together with several ISGs suggests a potential protective responsiveness of the ocular tissues to SARS-CoV-2.

Keywords: Cornea; Corneoscleral ring; Ocular surface; RNA-Sequencing; SARS-CoV-2.

Fig. 1

Principal Component Analysis based on whole transcriptome expression data. The samples labeled in blue are negative controls, whereas the ones labeled in red refer to positive donors. Triangles indicate samples negative or not-detected for SARS-CoV-2, whereas dots are samples with SARS-CoV-2 detected in the cornea and in the corneoscleral tissue (D1-D3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Volcano plot depicting gene expression and FDR-corrected p-values computed comparing PD-SARS-neg vs. CTRL samples (A), PD-SARS-pos vs. CRTL sample (B) and PD-SARS-neg vs. PD-SARS-pos samples (C). A selection of genes discussed in the present paper is highlighted by red dots their names are reported on the graphs.

Fig. 3

Venn diagram depicting exclusive and common DEGs in the comparisons between cluster1 (positive donors with undetected SARS-CoV-2), cluster 2 (positive donors with detected SARS-CoV-2) and controls (CTRL).