SARS-CoV-2 infection of ocular cells from human adult donor eyes and hESC-derived ocular lineages, Eriksen et al
The SARS-CoV-2 pandemic has resulted in an unparalleled disruption of global behavior and a significant loss of life. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible routes of entry is essential. While aerosol transmission is thought to be the primary route of spread, we assessed the eyes as an alternative point of entry. To this end, we confirmed expression of SARS-CoV-2 entry factors in post-mortem ocular surface tissue and productive viral replication in eye organoid cultures, most notably the limbus. Transcriptional analysis of ex vivo infected ocular surface cells and eye organoids revealed a robust induction of NF-κB in infected cells as well as diminished type-I/III interferon signaling. Together these data suggest that the eye can be directly infected by SARS-CoV-2 and implicates limbus as a portal for viral entry.
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Adult human eye tissue dissection and dissociation The eye tissues were separated from the eye globe using forceps and scissors then cut into 1mm2 pieces and placed into 0.25% collagenase (Worthington, NJ, USA), 3µg/ml DNase I Solution (STEMCELL Technologies) and 2µM Thiazovivin ROCK Inhibitor for a minimum of 2 hours. Cells were isolated from the tissue pieces by trituration, then pelleted by centrifugation at 356 x g for 5 min and plated into tissue culture treated plates coated with Synthemax II (Corning, AZ, USA). Cells were frozen down prior to single-cell RNA sequencing using CryoStor CS2 Freeze Media (Millipore Sigma) in Mr. Frosty containers. Virus SARS-related coronavirus 2 (SARS-CoV-2), isolate USA-WA1/2020 (NR-52281) was deposited by the Center for Disease Control and Prevention (Atlanta, GA, USA) and obtained through BEI Resources. The virus was propagated in Vero E6 cells in DMEM (GIBCO) supplemented with 2% FBS (Atlanta Biologicals), 4.5g/L D-glucose (GIBCO, 4mM L-glutamine (GIBCO), 10mM non-essential amino acids (GIBCO), 1mM sodium pyruvate (GIBCO), and 10mM HEPES (Fisher Scientific). Virus stock was washed twice in 15ml propagation medium to dilute out cytokines secreted into the culture medium during propagation by filtration through an Amicon Ultra-15 Centrifugal filter unit (100kDa, Millipore Sigma). Virus stock was resuspended in propagation medium. All infections were performed with the same passage 3 virus stock. Virus infections All experiments that involved live SARS-CoV-2 virus were carried out in a CDC/USDA-approved biosafety-level 3 facility at the Icahn School of Medicine at Mount Sinai (New York, USA) in accordance with institutional biosafety requirements. Virus stock was diluted in the appropriate growth medium for the specific cell type or SEAM culture with serum reduced to 2%. Cells were inoculated in a minimal volume for 1h at 37°C with agitation every 10 minutes to facilitate attachment. Inoculum was then removed, cells washed once in 1 x PBS and supplied with fresh appropriate culture medium with 2% serum. All infected samples were inactivated by a validated method before removal from the facility in accordance with institutional policies. RNA sequencing of viral infections 1µg of total RNA was enriched for polyadenylated RNA species and prepared for next-generation sequencing using the TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions. Sequencing libraries were sequenced on an Illumina NextSeq 500 platform. Bioinformatic analyses Raw reads were aligned to the human genome (hg19) using the RNA express app on Basespace (Illumina) and differential gene expression was determined using the DEseq2 protocol (Love et al., 2014). Raw reads were aligned to the SARS-CoV-2 genome (SARS-CoV-2/USA-WA1/2020 isolate, GenBank accession no. MN985325.1) using bowtie2 (Langmead and Salzberg, 2012) and read coverage was visualized using ggplot2 (Wickham, 2016).