In a recent study published on the Research Square preprint server* and under review in BMC Ophthalmology, researchers assessed the infectivity of two severe acute respiratory syndrome coronavirus lineage 2 (SARS-CoV-2) A variants in tissues human oculars in vitro.
Study: In vitro infection of human ocular tissues by SARS-CoV-2 lineage A isolates. Image credit: A Odo/Shutterstock
background
The main clinical presentation of coronavirus disease 2019 (COVID-19) is pneumonitis; however, studies have reported that ocular tissues may be susceptible to SARS-CoV-2 infections. In addition, in vitro studies have reported the presence of transmembrane protease serine 2 (TMPRSS2) and angiotensin-converting enzyme 2 (ACE2) in the corneal and conjunctival epithelium, which are essential for SARS entry -CoV-2 in the host. In addition, there are concerns about transmission of SARS-CoV-2 from corneas recovered from SARS-CoV-2-positive donors to corneal transplant recipients.
About the study
In the present study, the researchers assessed the susceptibility of human ocular tissues to SARS-CoV-2 infection by lineage A variants in vitro. They also evaluated the stability of SARS-CoV-2 A lineage variants in corneal preservation media.
Donor conjunctival, limbal, and corneal epithelia were cultured and inoculated with two variants of the SARS-CoV-2 A lineage belonging to the GISAID (Global Influenza Database Sharing Initiative) clade S, which they were: 2019-nCoV/USA-WA1/ 2020 (NR-52281) and isolate hCoV-19/Hong Kong/VM20001061/2020 (NR-52282). We assessed the susceptibility of the ocular surface to SARS-CoV-2 infections by the two isolates.
Donor eye tissues were obtained from donors with reports of negative tests for SARS-CoV-2 within ≤3 days before death or without SARS-CoV-2 infection based on the Eye Bank Association of America (EBAA) or the Food and Drug Administration (FDA) screening guidelines. In addition, Descemet stripping endothelial keratoplasty (DSAEK) grafts and cultured human corneal endothelial cells were treated with NR-52281 and NR-52282 to assess the susceptibility of the corneal endothelium to infection by SARS- CoV-2.
The samples were subjected to immunostaining analysis to assess the expression of SARS-CoV-2 spike protein (S) and nucleocapsid protein (N) to confirm SARS-CoV-2 infection. Subsequently, NR-52282 was inoculated into corneal preservation media and stored in cold storage (4oC) for two weeks, and assessed for alterations in SARS-CoV-2 titers. [tissue culture infectious dose (TCID50)]. Infection of Vero E6 cells (ATCC CRL-1586) was performed to confirm the infectivity of the two viral isolates and to propagate the viruses.
Results and discussion
In total, nine sets of conjunctival, limbal, and corneal epithelial cells were cultured from nine separate donor tissues, and three sets of endothelial cells were cultured for analysis. The median age of epithelial cell donors and graft donors was 78 and 68 years, respectively, and 67% and 50% of matched donors were male, respectively.
Isolates NR-52282 and NR-52281 caused extensive SARS-CoV-2 infections in Vero E6 cells two days post-infection (dpi) and infected cells showed SARS S and N expression -CoV-2 robust in immunostaining analysis. In contrast, there was no SARS-CoV-2 N expression in DSAEK graft keratocytes.
All three types of ocular epithelial cells were susceptible to SARS-CoV-2 infections caused by NR-52282 and NR-52281 at two dpi with 67% (n=16) and 68% (n=17) of specimens infected with isolate NR-52281 and NR-52282, respectively. Infection rates for conjunctival, corneal, and limbal epithelial cells were 78% (n=14), 65% (n=11), and 57% (n=8), respectively, and the rates of ‘infection were not significantly. differ between ocular epithelial cell types infected with any of the SARS-CoV-2 isolates.
After inoculation, the endothelium of four out of 10 DSAEK grafts (40%) had active SARS-CoV-2 infection caused by NR-52282. A <1-log decrease in SARS-CoV-2 titers was observed over two weeks in corneal preservation media. Samples from 22% (n = 2) of the donors were positive for SARS-CoV-2, and none of the donors showed complete resistance to infection across the inoculated samples. No statistically significant differences were observed in infection rates by sex and age.
Chondroitin sulfate (CS) is a substance present in corneal preservation media that helps stabilize human corneal endothelial membranes and may contribute to the stability of the SARS-CoV-2 envelope in the medium of corneal preservation, since SARS-CoV-2 is an enveloped ribonucleic acid. virus (RNA). Active SARS-CoV-2 infection and replication in donor corneas could result in higher titers of SARS-CoV-2; however, a marginal decrease in titers can be observed due to limitations in SARS-CoV-2 replication in cold storage and the presence of defensins.
Conclusions
Overall, the findings of the study demonstrated that SARS-CoV-2 lineage A variants could infect conjunctival, limbal, and corneal epithelium and corneal endothelium without statistically significant differences in the infectivity of lineage A variants In addition, contaminated donor corneas could have a higher risk of graft failure in the first days due to the replication of SARS-CoV-2 in the endothelial tissues.
In addition, SARS-CoV-2 lineage A isolates can remain stable and retain infectivity in corneal preservation media for two weeks at 4oC, precluding the use of transient quarantine measures to clear contaminated tissue. The study’s findings support ongoing screening of donor corneas for the presence of SARS-CoV-2 to minimize the risks of transplanting contaminated eye tissue.
*Important news
Research Square publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice or health-related behavior, or be treated as established information.