SARS-CoV-2 infects cells lining the blood-retinal barrier and induces a hyperinflammatory immune response in the retina via systemic exposure.

SARS-CoV-2 has been shown to cause wide-ranging ocular abnormalities and vision impairment in COVID-19 patients. However, there is limited understanding of SARS-CoV-2 in ocular transmission, tropism, and associated pathologies. The presence of viral RNA in corneal/conjunctival tissue and tears, along with the evidence of viral entry receptors on the ocular surface, has led to speculation that the eye may serve as a potential route of SARS-CoV-2 transmission. Here, we investigated the interaction of SARS-CoV-2 with cells lining the blood-retinal barrier (BRB) and the role of the eye in its transmission and tropism. The results from our study suggest that SARS-CoV-2 ocular exposure does not cause lung infection and moribund illness in K18-hACE2 mice despite the extended presence of viral remnants in various ocular tissues. In contrast, intranasal exposure not only resulted in SARS-CoV-2 spike (S) protein presence in different ocular tissues but also induces a hyperinflammatory immune response in the retina. Additionally, the long-term exposure to viral S-protein caused microaneurysm, retinal pigmented epithelium (RPE) mottling, retinal atrophy, and vein occlusion in mouse eyes. Notably, cells lining the BRB, the outer barrier, RPE, and the inner barrier, retinal vascular endothelium, were highly permissive to SARS-CoV-2 replication. Unexpectedly, primary human corneal epithelial cells were comparatively resistant to SARS-CoV-2 infection. The cells lining the BRB showed induced expression of viral entry receptors and increased susceptibility towards SARS-CoV-2-induced cell death. Furthermore, hyperglycemic conditions enhanced the viral entry receptor expression, infectivity, and susceptibility of SARS-CoV-2-induced cell death in the BRB cells, confirming the reported heightened pathological manifestations in comorbid populations. Collectively, our study provides the first evidence of SARS-CoV-2 ocular tropism via cells lining the BRB and that the virus can infect the retina via systemic permeation and induce retinal inflammation.

Evolution of ocular defects in infant macaques following in utero Zika virus infection.

Congenital Zika syndrome (CZS) is associated with microcephaly and various neurological, musculoskeletal, and ocular abnormalities, but the long-term pathogenesis and postnatal progression of ocular defects in infants are not well characterized. Rhesus macaques are superior to rodents as models of CZS because they are natural hosts of the virus and share similar immune and ocular characteristics, including blood-retinal barrier characteristics and the unique presence of a macula. Using a previously described model of CZS, we infected pregnant rhesus macaques with Zika virus (ZIKV) during the late first trimester and characterized postnatal ocular development and evolution of ocular defects in 2 infant macaques over 2 years. We found that one of them exhibited colobomatous chorioretinal atrophic lesions with macular and vascular dragging as well as retinal thinning caused by loss of retinal ganglion neuron and photoreceptor layers. Despite these congenital ocular malformations, axial elongation and retinal development in these infants progressed at normal rates compared with healthy animals. The ZIKV-exposed infants displayed a rapid loss of ZIKV-specific antibodies, suggesting the absence of viral replication after birth, and did not show any behavioral or neurological defects postnatally. Our findings suggest that ZIKV infection during early pregnancy can impact fetal retinal development and cause congenital ocular anomalies but does not appear to affect postnatal ocular growth.

Mechanisms of Blood-Retinal Barrier Disruption by HIV-1.

It has been found that human immunodeficiency virus (HIV)-1 RNA or antigens can be detected in the intraocular tissues of HIV-1 patients even under effective highly active anti-retroviral therapy (HAART). In vivo, blood-retinal barrier (BRB) establishes a critical, physiological guardian against microbial invasion of the eye, but may be compromised in the presence of HIV-1. The envelope glycoprotein gp120 is exposed on the surface of the HIV envelope, essential for virus entry into cells by the attachment to specific cell surface receptors. The BRB disruption by glycoprotein gp120 has been widely recognized, which is toxic to human retinal epithelial cells (RPE) and umbilical vein endothelial cells (HUVEC). The present review elaborates on various mechanisms of BRB disruption induced by HIV gp120, which may represent potential targets for the prevention of ocular HIV complications in the future.

Ocular Manifestations of Emerging Flaviviruses and the Blood-Retinal Barrier.

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.

Inflammation and outer blood-retina barrier (BRB) compromise following choroidal murine cytomegalovirus (MCMV) infections.

Purpose: The purpose of this study was to determine whether the blood-retina barrier is compromised by choroidal murine cytomegalovirus (MCMV) infection, using electron microscopy. Methods: BALB/c mice were immunosuppressed with methylprednisolone and monoclonal antibodies to CD4 and CD8. At several time points post-MCMV intraperitoneal inoculation, the eyes were removed and analyzed with western blotting and immunoelectron microscopy for the presence of MCMV early antigen (EA) and the host protein RIP3. Posterior eyecups from RIP3-/- and RIP3+/+ mice were cultured and inoculated with MCMV. At days 4, 7, and 11 post-infection, cultures were collected and analyzed with plaque assay, immunohistochemical staining, and real-time PCR (RT-PCR). Results: MCMV EA was observed in the nuclei of vascular endothelial cells and pericytes in the choriocapillaris. Disruption of Bruch's membrane was observed, especially at sites adjacent to activated platelets, and a few RPE cells containing some enlarged vesicles were found directly beneath disrupted Bruch's membrane. Some virus particles were also observed in the enlarged vesicles of RPE cells. Levels of the RIP3 protein, which was observed mainly in the RPE cells and the basement membrane of the choriocapillaris, were greatly increased following MCMV infection, while depletion of RIP3 resulted in greatly decreased inflammasome formation, as well as expression of downstream inflammation factors. Conclusions: The results suggest that systemic MCMV spreads to the choroid and replicates in vascular endothelia and pericytes of the choriocapillaris during immunosuppression. Choroidal MCMV infection is associated with in situ inflammation and subsequent disruption of Bruch's membrane and the outer blood-retina barrier.

Zika virus infection of cellular components of the blood-retinal barriers: implications for viral associated congenital ocular disease.

BACKGROUND: Ocular abnormalities present in microcephalic infants with presumed Zika virus (ZIKV) congenital disease includes focal pigment mottling of the retina, chorioretinal atrophy, optic nerve abnormalities, and lens dislocation. Target cells in the ocular compartment for ZIKV infectivity are unknown. The cellular response of ocular cells to ZIKV infection has not been described. Mechanisms for viral dissemination in the ocular compartment of ZIKV-infected infants and adults have not been reported. Here, we identify target cells for ZIKV infectivity in both the inner and outer blood-retinal barriers (IBRB and OBRB), describe the cytokine expression profile in the IBRB after ZIKV exposure, and propose a mechanism for viral dissemination in the retina. METHODS: We expose primary cellular components of the IBRB including human retinal microvascular endothelial cells, retinal pericytes, and Müller cells as well as retinal pigmented epithelial cells of the OBRB to the PRVABC56 strain of ZIKV. Viral infectivity was analyzed by microscopy, immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR and qRT-PCR). Angiogenic and proinflammatory cytokines were measured by Luminex assays. RESULTS: We find by immunofluorescent staining using the Flavivirus 4G2 monoclonal antibody that retinal endothelial cells and pericytes of the IBRB and retinal pigmented epithelial cells of the OBRB are fully permissive for ZIKV infection but not Müller cells when compared to mock-infected controls. We confirmed ZIKV infectivity in retinal endothelial cells, retinal pericytes, and retinal pigmented epithelial cells by RT-PCR and qRT-PCR using ZIKV-specific oligonucleotide primers. Expression profiles by Luminex assays in retinal endothelial cells infected with ZIKV revealed a marginal increase in levels of beta-2 microglobulin (ß2-m), granulocyte macrophage colony-stimulating factor (GMCSF), intercellular adhesion molecule 1 (ICAM-1), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP1), and vascular cell adhesion molecule 1 (VCAM-1) and higher levels of regulated upon activation, normal T cell expressed and presumably secreted (RANTES) but lower levels of interleukin-4 (IL-4) compared to controls. CONCLUSIONS: Retinal endothelial cells, retinal pericytes, and retinal pigmented epithelial cells are fully permissive for ZIKV lytic replication and are primary target cells in the retinal barriers for infection. ZIKV infection of retinal endothelial cells and retinal pericytes induces significantly higher levels of RANTES that likely contributes to ocular inflammation.

Zika virus infects cells lining the blood-retinal barrier and causes chorioretinal atrophy in mouse eyes.

Zika virus (ZIKV) is an important pathogen that causes not only neurologic, but also ocular, abnormalities. Thus, it is imperative that models to study ZIKV pathogenesis in the eye are developed to identify potential targets for interventions. Here, we studied ZIKV interactions with human retinal cells and evaluated ZIKV's pathobiology in mouse eyes. We showed that cells lining the blood-retinal barrier (BRB), the retinal endothelium, and retinal pigment epithelium (RPE) were highly permissive and susceptible to ZIKV-induced cell death. Direct inoculation of ZIKV in eyes of adult C57BL/6 and IFN-stimulated gene 15 (ISG15) KO mice caused chorioretinal atrophy with RPE mottling, a common ocular manifestation of congenital ZIKV infection in humans. This response was associated with induced expression of multiple inflammatory and antiviral (IFNs) response genes in the infected mouse retina. Interestingly, ISG15 KO eyes exhibited severe chorioretinitis, which coincided with increased retinal cell death and higher ZIKV replication. Collectively, our study provides the first evidence to our knowledge that ZIKV causes retinal lesions and infects the cells lining the BRB and that ISG15 plays a role in retinal innate defense against ZIKV infection. Our mouse model can be used to study mechanisms underlying ZIKV-induced chorioretinitis and to gauge ocular antiviral therapies.

HIV-1 gp120 Glycoprotein Interacting with Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Non-integrin (DC-SIGN) Down-Regulates Tight Junction Proteins to Disrupt the Blood Retinal Barrier and Increase Its Permeability.

Approximately 70% of HIV-1 infected patients acquire ocular opportunistic infections and manifest eye disorders during the course of their illness. The mechanisms by which pathogens invade the ocular site, however, are unclear. Under normal circumstances, vascular endothelium and retinal pigment epithelium (RPE), which possess a well developed tight junction complex, form the blood-retinal barrier (BRB) to prevent pathogen invasion. We hypothesize that disruption of the BRB allows pathogen entry into ocular sites. The hypothesis was tested using in vitro models. We discovered that human RPE cells could bind to either HIV-1 gp120 glycoproteins or HIV-1 viral particles. Furthermore, the binding was mediated by dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expressed on RPE cells. Upon gp120 binding to DC-SIGN, cellular NF-κB signaling was triggered, leading to the induction of matrix metalloproteinases, which subsequently degraded tight junction proteins and disrupted the BRB integrity. DC-SIGN knockdown or prior blocking with a specific antibody abolished gp120-induced matrix metalloproteinase expression and reduced the degradation of tight junction proteins. This study elucidates a novel mechanism by which HIV, type 1 invades ocular tissues and provides additional insights into the translocation or invasion process of ocular complication-associated pathogens.

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease.

Müller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling Müller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms. There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5 has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label Müller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

Intraocular and plasma HIV-1 RNA loads and HIV uveitis.

OBJECTIVE: The objective of this study was to analyze human immunodeficiency virus (HIV) dynamics across the blood-retinal barrier and to determine whether the high levels of HIV in the eye are associated with any ocular disorders in HIV-infected patients. DESIGN: This study included a prospective case series of 40 HIV-positive patients with uveitis. INTERVENTION: Clinical and laboratory examinations included plasma and intraocular HIV-1 RNA loads as well as the clinical manifestations of uveitis. RESULTS: Intraocular HIV-1 RNA was detected in 32% (13/40) of HIV-positive patients with uveitis. Intraocular HIV-1 RNA loads were associated with high HIV-1 RNA plasma loads (P < 0.001) and not being on HAART therapy (P = 0.005). In addition, detectable intraocular HIV-1 RNA levels were higher in patients with the absence of retinal lesions (P = 0.008). In three patients, the HIV load in the eye largely exceeded that of plasma. These three patients had all bilateral anterior uveitis and/or vitritis without retinal lesions and exhibited no evidence of other intraocular infectious agents causing uveitis than HIV itself. CONCLUSION: The eye can form a sanctuary where HIV might replicate and cause an inflammatory reaction.

Supernatants from human cytomegalovirus (HCMV)-infected retinal glial cells increase transepithelial electrical resistance in a cell culture model: evidence of HCMV immune escape in the eye?

The underlying mechanisms leading to persistence of human cytomegalovirus (HCMV) in the immune privileged retina are not fully understood. This in vitro study was done to evaluate the influence of HCMV-infected retinal glial cells on epithelial barrier functions. Glial cells derived from human eyes were cultured and infected with the clinical HCMV isolate Hi91. Supernatants of mock (GS(mock)) and Hi91 (GS(Hi91)) -infected glial cells were collected at 72 h post inoculation and used for incubation of CaCo-2 cells grown in transwell chambers. Transepithelial electrical resistance (TER) was analyzed as a measure of epithelial integrity. Virus-free GS(Hi91 )but not GS(mock) increased TER from 250 Omega/cm(2) to more than 1,000 Omega/cm(2)within 2 h. Increased TER values were measured up to 48 h (n = 3). No changes in TER were observed when conditioned supernatants from HCMV-infected human foreskin fibroblasts were used. No evidence of GS(Hi91)-induced modification of beta-catenin (zonula adherens) or occludin and ZO-1 (zonula occludens) was found. Our results suggest that HCMV-infected glial cells may support epithelial barrier functions by a yet unknown mechanism. Our findings may help to explain the ocular persistence of HCMV and the maintenance of ocular immune privilege early in infection.