Background SARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues. Methods The expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti -SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in 10 COVID-19 donors. Findings Of 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ∼13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti -SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP–I) disinfection. Interpretations Our study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation.

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Viruses are known to perturb host cellular metabolism to enable their replication and spread. However, little is known about the interactions between Zika virus (ZIKV) infection and host metabolism. Using primary human retinal vascular endothelial cells and an established human endothelial cell line, we investigated the role of AMP-activated protein kinase (AMPK), a master regulator of energy metabolism, in response to ZIKV challenge. ZIKV infection caused a time-dependent reduction in the active phosphorylated state of AMPK and of its downstream target acetyl-CoA carboxylase. Pharmacological activation of AMPK using 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), metformin, and a specific AMPKa activator (GSK621) attenuated ZIKV replication. This activity was reversed by an AMPK inhibitor (compound C). Lentivirus-mediated knockdown of AMPK and the use of AMPKa 2/2 mouse embryonic fibroblasts provided further evidence that AMPK has an antiviral effect on ZIKV replication. Consistent with its antiviral effect, AMPK activation potentiated the expression of genes with antiviral properties (e.g., IFNs, OAS2, ISG15, and MX1) and inhibited inflammatory mediators (e.g., TNF-a and CCL5). Bioenergetic analysis showed that ZIKV infection evokes a glycolytic response, as evidenced by elevated extracellular acidification rate and increased expression of key glycolytic genes (GLUT1, HK2, TPI, and MCT4); activation of AMPK by AICAR treatment reduced this response. Consistent with this, 2-deoxyglucose, an inhibitor of glycolysis, augmented AMPK activity and attenuated ZIKV replication. Thus, our study demonstrates that the anti-ZIKV effect of AMPK signaling in endothelial cells is mediated by reduction of viral-induced glycolysis and enhanced innate antiviral responses.

Despite flaviviruses remaining the leading cause of systemic human infections worldwide, ocular manifestations of these mosquito-transmitted viruses are considered relatively uncommon in part due to under-reporting. However, recent outbreaks of Zika virus (ZIKV) implicated in causing multiple ocular abnormalities, such as conjunctivitis, retinal hemorrhages, chorioretinal atrophy, posterior uveitis, optic neuritis, and maculopathies, has rejuvenated a significant interest in understanding the pathogenesis of flaviviruses, including ZIKV, in the eye. In this review, first, we summarize the current knowledge of the major flaviviruses (Dengue, West Nile, Yellow Fever, and Japanese Encephalitis) reported to cause ocular manifestations in humans with emphasis on recent ZIKV outbreaks. Second, being an immune privilege organ, the eye is protected from systemic infections by the presence of blood-retinal barriers (BRB). Hence, we discuss how flaviviruses modulate retinal innate response and breach the protective BRB to cause ocular or retinal pathology. Finally, we describe recently identified infection signatures of ZIKV and discuss whether these system biology-predicted genes or signaling pathways (e.g., cellular metabolism) could contribute to the pathogenesis of ocular manifestations and assist in the development of ocular antiviral therapies against ZIKV and other flaviviruses.

Purpose: Zika virus (ZIKV) has emerged as an important human pathogen causing ocular complications. There have been reports of the shedding of ZIKV in human as well as animal tears. In this study, we investigated the infectivity of ZIKV in corneal epithelial cells and their antiviral immune response. Methods: Primary human corneal epithelial cells (Pr. HCECs) and an immortalized cell line (HUCL) were infected with two different strains of ZIKV (PRVABC59 & BeH823339) or dengue virus (DENV, serotypes 1-4). Viral infectivity was assessed by immunostaining of viral antigen and plaque assay. qRT-PCR and immunoblot analyses were used to assess the expression of innate inflammatory and antiviral genes. Supplementation of recombinant ISG15 (rISG15) and gene silencing approaches were used to elucidate the role of ISG15 in corneal antiviral defense. Results: Pr. HCECs, but not the HUCL cells, were permissive to both ZIKV strains and specifically to DENV3 infection. ZIKV induced the expression of viral recognition receptors (TLR3, RIG-I, & MDA5), and genes involved in inflammatory (CXCL10& CCL5) and antiviral (IFNs, MX1, OAS2, ISG15) responses in Pr. HCECs. Furthermore, ZIKV infection caused Pr. HCECs cell death, as evidenced by TUNEL staining. Silencing of ISG15 increased ZIKV infectivity while supplementation with rISG15 reduced ZIKV infection by direct inactivation of ZIKV and inhibiting its entry. Conclusions: Our study demonstrates for the first time, that ZIKV virus can readily infect and replicate in Pr. HCECs. Therefore, ZIKV may persist in the cornea and pose the potential risk of transmission via corneal transplantation.

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