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Purpose: The purpose of this study was to investigate whether corneal lesions in mice with type 2 diabetes mellitus (T2D) infected with herpes simplex virus (HSV)-1 are more severe, and to elucidate the specific underlying mechanism., Methods: The corneas of control mice and T2D mice induced by a high-fat diet combined with streptozotocin were infected with the HSV-1 Mckrae strain to assess corneal infection, opacity, and HSV-1 replication. RNA sequencing of the corneal epithelium from wild-type and db/db mice (a genetic T2D mouse model) was conducted to identify the key gene affecting T2D infection. Immunofluorescence staining was performed on corneal sections from T2D mice and patients with T2D. The effect of small interfering RNA (siRNA) knockdown on corneal HSV-1 infection was evaluated in both in vitro and in vivo models., Results: T2D mice exhibited a more severe infection phenotype following HSV-1 infection, characterized by augmented corneal opacity scores, elevated viral titers, and transcripts compared to control mice. Transcriptome analysis of corneal epithelium revealed a hyperactive viral response in T2D mice, highlighting the differentially expressed gene Rtp4 (encoding receptor transporter protein 4). Receptor transporter protein 4 (RTP4) expression was enhanced in the corneal epithelium of T2D mice and patients with T2D. Virus binding assays demonstrated that RTP4 facilitated HSV-1 binding to human corneal epithelial cells. Silencing RTP4 alleviated HSV-1 infection in both in vitro and in vivo T2D models., Conclusions: The findings indicate that elevated RTP4 exacerbates HSV-1 infection by enhancing its binding to corneal epithelial cells, whereas Rtp4 knockdown mitigated corneal lesions in T2D mice. This implies RTP4 as a potential target for intervention in diabetic HSV-1 infection.

Herpes stromal keratitis is the leading cause of infectious blindness in the western world. Infection by HSV1 is most common, but VZV and hCMV also infect the cornea. Multiple models of HSV1 corneal infection exist, but none for VZV and hCMV because of their host specificity. Here, we used commercially available 3D human corneal epithelial equivalents (HCEE) to study infection by these herpesviruses. HCEE was infected by HSV-1 and hCMV without requiring scarification and resulted in spreading infections. Spread of HSV-1 infection was rapid, while that of hCMV was slow. In contrast, infections with VZV required damage to the HCEE and did not spread. Acyclovir dramatically reduced replication of HSV-1 in this model. We conclude that highly quality-controlled, readily available HCEE is a useful model to study human-restricted herpesvirus infection of the human corneal epithelium and for screening of antiviral drugs for treating HSK in an 3D model system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Herpes simplex keratitis (HSK) is the most common but serious infectious keratitis with high recurrence. It is predominantly caused by herpes simplex virus type 1 (HSV-1). The spread mechanism of HSV-1 in HSK is not entirely clear. Multiple publications indicate that exosomes participate in the intercellular communication process during viral infections. However, there is rare evidence that HSV-1 spreads in HSK by exosomal pathway. This study aims to investigate the relationship between the spread of HSV-1 and tear exosomes in recurrent HSK., Methods: Tear fluids collected from total 59 participants were included in this study. Tear exosomes were isolated by ultracentrifugation, then identified by silver staining and western blot. The size was determined by dynamic light scattering (DLS). The viral biomarkers were identified by western blot. The cellular uptake of exosomes was studied using labelled exosomes., Results: Tear exosomes were indeed enriched in tear fluids. Collected exosomes own normal diameters consistent with related reports. The exosomal biomarkers existed in tear exosomes. Labelled exosomes were successfully taken up by human corneal epithelial cells (HCEC) in large numbers in a short time. After cellular uptake, HSK biomarkers were detectable by western blot in infected cells., Conclusions: Tear exosomes should be the latent sites of HSV-1 in recurrent HSK and might be involved in the spread of HSV-1. Besides, this study verifies HSV-1 genes can be indeed transferred between cells by exosomal pathway, providing new inspiration for the clinical intervention and treatment as well as the drug discovery of recurrent HSK., (© 2023. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Coronavirus disease 2019 (COVID‑19), caused by the severe acute respiratory syndrome coronavirus‑2 (SARS‑CoV‑2), led to an outbreak of viral pneumonia in December 2019. The present study aimed to investigate the host inflammatory response signature‑caused by SARS‑CoV‑2 in human corneal epithelial cells (HCECs). The expression level of angiotensin‑converting enzyme 2 (ACE2) in the human cornea was determined via immunofluorescence. In vitro experiments were performed in HCECs stimulated with the SARS‑CoV‑2 spike protein. Moreover, the expression levels of ACE2, IL‑8, TNF‑α, IL‑6, gasdermin D (GSDMD) and IL‑1β in HCECs were detected using reverse transcription‑quantitative PCR and/or western blotting. It was identified that ACE2 was expressed in normal human corneal epithelium and HCECs cultured in vitro . Furthermore, the expression levels of IL‑8, TNF‑α and IL‑6 in HCECs were decreased following SARS‑CoV‑2 spike protein stimulation, while the expression levels of GSDMD and IL‑1β were increased. In conclusion, the present results demonstrated that the SARS‑CoV‑2 spike protein suppressed the host inflammatory response and induced pyroptosis in HCECs. Therefore, blocking the ACE2 receptor in HCECs may reduce the infection rate of COVID‑19.

Purpose: To compare efficiency and tolerance between topical 0.5% cyclosporine A (CSA) and fluorometholone (FML) for subepithelial infiltrates (SEI) complicating epidemic keratoconjunctivitis., Methods: We conducted a prospective double-blind randomized study involving 72 eyes with SEI. Thirty-eight eyes were treated with topical FML (FML group) and 34 eyes with CSA 0.5% eye drops (CSA group). Treatment was considered successful in case of SEI reduction and visual acuity improvement. Tolerance was evaluated by Schirmer test value, burning on eye drops instillation, and conjunctival injection., Results: Baseline characteristics of both groups were similar (P > 0.05). After 3 months of the regimen, resolution of SEI was 3 times more observed in the FML group than that in the CSA group (P = 0.026). After 6 months, resolution of SEI was observed in 70% of the FML group and in 47% of the CSA group (P = 0.068). The recurrence of SEI was almost twice higher in the FML group than that in the CSA group (16% vs. 9%). FML was better tolerated during the first 3 months: a higher Schirmer test value (P = 0.0003), less burning on instillation (P = 0.242), and less conjunctival injection (P = 0.003). For the rest of the follow-up period, the 2 groups were comparable in tolerance. No ocular hypertension was noted., Conclusions: Epidemic keratoconjunctivitis can evolve favorably under both FML and CSA. The effect of FML is faster and CSA is more durable with fewer recurrences. Both are safe therapeutic options for long-term control of SEI., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: The high infection rate of SARS-CoV-2 necessitates the need for multiple studies identifying the molecular mechanisms that facilitate the viral entry and propagation. Currently the potential extra-respiratory transmission routes of SARS-CoV-2 remain unclear., Methods: Using single-cell RNA Seq and ATAC-Seq datasets and immunohistochemical analysis, we investigated SARS-CoV-2 tropism in the embryonic, fetal and adult human ocular surface., Results: The co-expression of ACE2 receptor and entry protease TMPRSS2 was detected in the human adult conjunctival, limbal and corneal epithelium, but not in the embryonic and fetal ocular surface up to 21 post conception weeks. These expression patterns were corroborated by the single cell ATAC-Seq data, which revealed a permissive chromatin in ACE2 and TMPRSS2 loci in the adult conjunctival, limbal and corneal epithelium. Co-expression of ACE2 and TMPRSS2 was strongly detected in the superficial limbal, corneal and conjunctival epithelium, implicating these as target entry cells for SARS-CoV-2 in the ocular surface. Strikingly, we also identified the key pro-inflammatory signals TNF, NFKβ and IFNG as upstream regulators of the transcriptional profile of ACE2 + TMPRSS2 + cells in the superficial conjunctival epithelium, suggesting that SARS-CoV-2 may utilise inflammatory driven upregulation of ACE2 and TMPRSS2 expression to enhance infection in ocular surface., Conclusions: Together our data indicate that the human ocular surface epithelium provides an additional entry portal for SARS-CoV-2, which may exploit inflammatory driven upregulation of ACE2 and TMPRSS2 entry factors to enhance infection., (Copyright © 2020. Published by Elsevier Inc.)

Purpose: Ocular infection by human adenovirus species D type 37 (HAdV-D37) causes epidemic keratoconjunctivitis, a severe, hyperacute condition. The corneal component of epidemic keratoconjunctivitis begins upon infection of corneal epithelium, and the mechanism of viral entry dictates subsequent proinflammatory gene expression. Therefore, it is important to understand the specific pathways of adenoviral entry in these cells., Methods: Transmission electron microscopy of primary and tert-immortalized human corneal epithelial cells infected with HAdV-D37 was performed to identify the means of viral entry. Confocal microscopy was used to determine intracellular trafficking. The results of targeted small interfering RNA and specific chemical inhibitors were analyzed by quantitative PCR, and Western blot., Results: By transmission electron microscopy, HAdV-D37 was seen to enter by both clathrin-coated pits and macropinocytosis; however, entry was both pH and dynamin 2 independent. Small interfering RNA against clathrin, AP2A1, and lysosome-associated membrane protein 1, but not early endosome antigen 1, decreased early viral gene expression. Ethyl-isopropyl amiloride, which blocks micropinocytosis, did not affect HAdV-D37 entry, but IPA, an inhibitor of p21-activated kinase, and important to actin polymerization, decreased viral entry in a dose-dependent manner., Conclusions: HAdV-D37 enters human corneal epithelial cells by a noncanonical clathrin-mediated pathway involving lysosome-associated membrane protein 1 and PAK1, independent of pH, dynamin, and early endosome antigen 1. We showed earlier that HAdV-D37 enters human keratocytes through caveolae. Therefore, epidemic keratoconjunctivitis-associated viruses enter different corneal cell types via disparate pathways, which could account for a relative paucity of proinflammatory gene expression upon infection of corneal epithelial cells compared with keratocytes, as seen in prior studies.

Purpose: Herpes simplex virus type I (HSV-1) infection of corneal epithelial cells activates ataxia telangiectasia mutated (ATM), an apical kinase in the host DNA damage response pathway, whose activity is necessary for the progression of lytic HSV-1 infection. The purpose of this study is to investigate the mechanism of ATM activation by HSV-1 in the corneal epithelium, as well as its functional significance., Methods: Mechanistic studies were performed in cultured human corneal epithelial cell lines (hTCEpi, HCE), as well as in esophageal (EPC2) and oral (OKF6) cell lines. Transfection-based experiments were performed in HEK293 cells. HSV-1 infection was carried out using the wild-type KOS strain, various mutant strains (tsB7, d120, 7134, i13, n208), and bacterial artificial chromosomes (fHSVΔpac, pM24). Inhibitors of ATM (KU-55933), protein synthesis (cycloheximide), and viral DNA replication (phosphonoacetic acid) were used. Outcomes of infection were assayed using Western blotting, qRT-PCR, immunofluorescence, and comet assay., Results: This study demonstrates that HSV-1-mediated ATM activation in corneal epithelial cells relies on the viral immediate early gene product ICP4 and requires the presence of the viral genome in the host nucleus. We show that ATM activation is independent of viral genome replication, the ICP0 protein, and the presence of DNA lesions. Interestingly, ATM activity appears to be necessary at the onset of infection, but dispensable at the later stages., Conclusions: This study expands our understanding of HSV-1 virus-host interactions in the corneal epithelium and identifies potential areas of future investigation and therapeutic intervention in herpes keratitis.

This retrospective observational study aims to report the clinical characteristics and surgical results in eyes with Posner-Schlossman syndrome (PSS), and compare these outcomes between cytomegalovirus (CMV)-positive and -negative eyes.We reviewed the medical records of 21 consecutive immunocompetent patients clinically diagnosed with PSS between the years 2010 and 2018. Aqueous humor was collected from all the affected eyes to detect if CMV was present, and polymerase chain reaction (PCR) was performed using the herpesvirus family primers.The average period between the initial PSS attack and aqueous humor sampling at our institute was 9.3 years. Out of the 21 patients, 62% were CMV-positive. Regardless of CMV status, the mean intraocular pressure (IOP), mean deviation (MD), and central corneal endothelium cell (CEC) density, at the initial examination at our institute were already significantly worse in the affected eyes than in the unaffected eyes (all P values < .05). The average visual acuity (VA) was only significantly worse in the CMV-positive group (P = .02). Out of all the patients, those that were CMV-positive had undergone more glaucoma surgeries (P = .056). Fourteen patients underwent either a trabeculectomy (TRAB) or a trabeculotomy (LOT), and their IOP significantly reduced following surgery (P < .001). In 85.7% of those that had surgery, their IOP was successfully lowered to less than 20 mm Hg.Long-lasting PSS causes a decrease in VA, MD, and the CEC density. A prompt diagnosis is required, and an appropriate treatment plan should be formulated. In those patients with PSS that develop uncontrolled glaucoma, both TRAB and LOT may be effective in controlling IOP.

HSV-1 infection in corneal epithelium initiates the process of herpes simplex keratitis. We investigated the dynamic change of the host proteins in corneal epithelial cells infected with HSV-1 to understand the virus-host interaction. iTRAQ coupled with LC-MS/MS was applied to quantitatively analyze the protein profiles in HSV-1 infected corneal epithelial cells at 6 and 24 h post-infection (hpi), and the results were validated by multiple reaction monitoring (MRM). We also performed bioinformatic analysis to investigate the potentially important signal pathways and protein interaction networks in the host response to HSV-1 infection. We identified 292 proteins were up-regulated and 168 proteins were down-regulated at 6 hpi, while 132 proteins were up-regulated and 89 proteins were down-regulated at 24 hpi, which were validated by MRM analysis. We found the most enriched GO terms were translational initiation, cytosol, poly(A) RNA binding, mRNA splicing via spliceosome and extracellular exosome for the dysregulated proteins. KEGG pathway analysis revealed significant changes in metabolism pathway characterized by decreased tricarboxylic acid cycle activity and increased glycolysis. Proteins interaction network analysis indicated several proteins including P4HB, ACLY, HSP90AA1 and EIF4A3, might be critical proteins in the host-virus response. Our study for the first time analyzed the protein profile of HSV-1 infected primary corneal epithelial cells by quantitative proteomics. These findings help to better understand the host-virus interaction and the pathogenesis of herpes simplex keratitis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Herpes simplex virus 1 (HSV-1) cycles between phases of latency in sensory neurons and replication in mucosal sites. HSV-1 encodes two key proteins that antagonize the shutdown of host translation, US11 through preventing PKR activation and ICP34.5 through mediating dephosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). While profound attenuation of ICP34.5 deletion mutants has been repeatedly demonstrated, a role for US11 in HSV-1 pathogenesis remains unclear. We therefore generated an HSV-1 strain 17 US11-null virus and examined its properties in vitro and in vivo In U373 glioblastoma cells, US11 cooperated with ICP34.5 to prevent eIF2α phosphorylation late in infection. However, the effect was muted in human corneal epithelial cells (HCLEs), which did not accumulate phosphorylated eIF2α unless both US11 and ICP34.5 were absent. Low levels of phosphorylated eIF2α correlated with continued protein synthesis and with the ability of virus lacking US11 to overcome antiviral immunity in HCLE and U373 cells. Neurovirulence following intracerebral inoculation of mice was not affected by the deletion of US11. In contrast, the time to endpoint criteria following corneal infection was greater for the US11-null virus than for the wild-type virus. Replication in trigeminal ganglia and periocular tissue was promoted by US11, as was periocular disease. The establishment of latency and the frequency of virus reactivation from trigeminal ganglia were unaffected by US11 deletion, although emergence of the US11-null virus occurred with slowed kinetics. Considered together, the data indicate that US11 facilitates the countering of antiviral response of infected cells and promotes the efficient emergence of virus following reactivation. IMPORTANCE Alphaherpesviruses are ubiquitous DNA viruses and include the human pathogens herpes simplex virus 1 (HSV-1) and HSV-2 and are significant causes of ulcerative mucosal sores, infectious blindness, encephalitis, and devastating neonatal disease. Successful primary infection and persistent coexistence with host immune defenses are dependent on the ability of these viruses to counter the antiviral response. HSV-1 and HSV-2 and other primate viruses within the Simplexvirus genus encode US11, an immune antagonist that promotes virus production by preventing shutdown of protein translation. Here we investigated the impact of US11 deletion on HSV-1 growth in vitro and pathogenesis in vivo This work supports a role for US11 in pathogenesis and emergence from latency, elucidating immunomodulation by this medically important cohort of viruses., (Copyright © 2019 American Society for Microbiology.)

Glycans on plasma membranes and in secretions play important roles in infection by many viruses. Species D human adenovirus type 37 (HAdV-D37) is a major cause of epidemic keratoconjunctivitis (EKC) and infects target cells by interacting with sialic acid (SA)-containing glycans via the fiber knob domain of the viral fiber protein. HAdV-D37 also interacts with sulfated glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, we investigated the molecular requirements of HAdV-D37 fiber knob:GAG interactions using a GAG microarray and demonstrated that fiber knob interacts with a broad range of sulfated GAGs. These interactions were corroborated in cell-based assays and by surface plasmon resonance analysis. Removal of heparan sulfate (HS) and sulfate groups from human corneal epithelial (HCE) cells by heparinase III and sodium chlorate treatments, respectively, reduced HAdV-D37 binding to cells. Remarkably, removal of HS by heparinase III enhanced the virus infection. Our results suggest that interaction of HAdV-D37 with sulfated GAGs in secretions and on plasma membranes prevents/delays the virus binding to SA-containing receptors and inhibits subsequent infection. We also found abundant HS in the basement membrane of the human corneal epithelium, which may act as a barrier to sub-epithelial infection. Collectively, our findings provide novel insights into the role of GAGs as viral decoy receptors and highlight the therapeutic potential of GAGs and/or GAG-mimetics in HAdV-D37 infection.

Purpose: While VZV DNA and antigen have been detected in acute and chronic VZV keratitis, it is unclear whether productive infection of corneal cells is ongoing or whether residual, noninfectious VZV antigens elicit inflammation. Herein, we examined VZV-infected primary human corneal epithelial cells (HCECs) and keratocytes (HKs) to elucidate the pathogenesis of VZV keratitis., Methods: HCECs and HKs were mock- or VZV infected. Seven days later, cells were examined for morphology, proinflammatory cytokine and matrix metalloproteinase (MMP) release, ability to recruit peripheral blood mononuclear cells (PBMCs) and neutrophils, and MMP substrate cleavage., Results: Both cell types synthesized infectious virus. VZV-infected HCECs proliferated, whereas VZV-infected HKs died. Compared to mock-infected cells, VZV-infected HCECs secreted significantly more IL-6, IL-8, IL-10, and IL-12p70 that were confirmed at the transcript level, and MMP-1 and MMP-9; conditioned supernatant attracted PBMCs and neutrophils and cleaved MMP substrates. In contrast, VZV-infected HKs suppressed cytokine secretion except for IL-8, which attracted neutrophils, and suppressed MMP release and substrate cleavage., Conclusions: Overall, VZV-infected HCECs recapitulate findings of VZV keratitis with respect to epithelial cell proliferation, pseudodendrite formation and creation of a proinflammatory environment, providing an in vitro model for VZV infection of corneal epithelial cells. Furthermore, the proliferation and persistence of VZV-infected HCECs suggest that these cells may serve as viral reservoirs if immune clearance is incomplete. Finally, the finding that VZV-infected HKs die and suppress most proinflammatory cytokines and MMPs may explain the widespread death of these cells with unchecked viral spread due to ineffective recruitment of PBMCs.

Purpose: Long noncoding RNAs (lncRNAs) have been demonstrated to have important regulatory functions in diverse cellular processes; however, the role of lncRNAs in the pathogenesis of herpes simplex keratitis (HSK) remains poorly understood., Methods: Primary human corneal epithelial cells (HCECs) were infected with herpes simplex virus-1 (HSV-1) and the total RNAs extracted from both the infected group and the mock-infected group subjected to microarray analysis to identify the differential expression of lncRNAs and mRNAs. We also performed bioinformatic analysis including gene ontology (GO) analysis, pathway analysis and co-expression network analysis., Results: Compared with mock-infected group, the expression of thousands of lncRNAs and mRNAs were significantly changed, and the microarray results were validated by qRT-PCR. The most enriched GOs targeted by up-regulated transcripts were defense response, intrinsic component of plasma membrane and cytokine activity，and the most enriched GOs targeted by the down-regulated transcripts were cellular metabolic process, intracellular part and poly (A) RNA binding. Pathway analysis indicated that the most correlated pathways for up- and down-regulated transcripts were cytokine-cytokine receptor interaction and RNA transport, respectively., Conclusions: Our study identified the genome-wide profile of lncRNAs and mRNAs expression in primary corneal epithelial cells with HSV-1 infection. These transcriptomic data together with subsequent bioinformatic analysis will provide us with novel clue to the insight into molecular mechanism and potential therapeutic targets of HSK. Further studies are expected to verify the potentially functional genes and pathways and explore the critical lncRNAs., Abbreviations: Long noncoding RNAs: lncRNAs; herpes simplex virus-1: HSV-1; herpes simplex virus keratitis: HSK; human corneal epithelial cells: HCECs.

Immune privilege helps protect the cornea from damaging inflammation but can also impair pathogen clearance from this mucosal surface. Programmed death-ligand 1 (PD-L1 or B7-H1) contributes to corneal immune privilege by inhibiting the function of a variety of immune cells. We asked whether programmed death-1 (PD-1)/PD-L1 interaction regulates HSV-1 clearance from infected corneas. We show that PD-L1 is constitutively expressed in the corneal epithelium and is upregulated upon HSV-1 corneal infection, with peak expression on CD45 + cells NK cells, dendritic cells, neutrophils, and macrophages and CD45 - corneal epithelial cells at 4 d postinfection (dpi). As early as 1 dpi, HSV-1-infected corneas of B7-H1 -/- mice as compared with wild-type mice showed increased chemokine expression and this correlated with increased migration of inflammatory cells into the viral lesions and decreased HSV-1 corneal titers. Local PD-L1 blockade caused a similar increase in viral clearance, suggesting a local effect of PD-1/PD-L1 in the cornea. The enhanced HSV-1 clearance at 2 dpi resulting from PD-1/PD-L1 blockade is mediated primarily by a monocyte/macrophage population. Studies in bone marrow chimeras demonstrated enhanced viral clearance when PD-L1 was absent only from nonhematopoietic cells. We conclude that PD-L1 expression on corneal cells negatively impacts the ability of the innate immune system to clear HSV-1 from infected corneas., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Herpes simplex virus type 1 (HSV-1) causes recurrent mucocutaneous lesions in the eye that may advance to corneal blindness. Nucleoside analogs exemplified by acyclovir (ACV) form the primary class of antiherpetic drugs, but this class suffers limitations due to the emergence of viral resistance and other side effects. While studying the molecular basis of ocular HSV-1 infection, we observed that BX795, a commonly used inhibitor of TANK-binding kinase 1 (TBK1), strongly suppressed infection by multiple strains of HSV-1 in transformed and primary human cells, cultured human and animal corneas, and a murine model of ocular infection. Our investigations revealed that the antiviral activity of BX795 relies on targeting Akt phosphorylation in infected cells, leading to the blockage of viral protein synthesis. This small-molecule inhibitor, which was also effective against an ACV-resistant HSV-1 strain, shows promise as an alternative to existing drugs and as an effective topical therapy for ocular herpes infection. Collectively, our results obtained using multiple infection models and virus strains establish BX795 as a promising lead compound for broad-spectrum antiviral applications in humans., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose: To evaluate the therapeutic benefit of self-retained cryopreserved amniotic membrane in conjunction with oral antiviral therapy in herpetic epithelial keratitis., Methods: Retrospective review of 4 patients with primary (1 eye) and recurrent (3 eyes) unilateral herpetic epithelial keratitis treated with cryopreserved amniotic membrane through the placement of the PROKERA Slim (PKS) (Bio-Tissue, Inc) in conjunction with oral acyclovir. Their symptoms, conjunctival inflammation, corneal staining, and visual acuity were compared before and after treatment., Results: Herpetic epithelial keratitis presented as dendritic (3 eyes) and geographic (1 eye) epithelial lesions. After epithelial debridement and placement of the PKS for 5 ± 3.7 days, all patients reported significant relief of symptoms, rapid corneal epithelialization, and reduction of ocular surface inflammation. The visual acuity was also improved in all eyes from 0.7 ± 0.7 to 0.4 ± 0.7 logarithm of the minimum angle of resolution (P = 0.2). They remained symptom-free during a follow-up period of 2.7 to 50.8 (20.3 ± 21.7) months., Conclusions: The PKS in conjunction with oral acyclovir facilitates the ease of early intervention to accelerate restoration of a normal corneal epithelium in herpetic epithelial keratitis.

Purpose: Infection of the human cornea by herpes simplex virus type-1 (HSV-1) can cause significant vision loss. The purpose of this study was to develop an ex vivo model to visualize viral growth and spread in the cornea. The model was also used to analyze cytokine production and study the antiviral effects of zinc oxide tetrapods., Methods: A β-galactosidase-expressing recombinant virus, HSV-1(KOS)tk12, was used to demonstrate the ability of the virus to enter and develop blue plaques on human corneal epithelial (HCE) cells and corneal tissues. Freshly obtained porcine corneas were cultured and then scratched before infection with HSV-1(KOS)tk12. The blue plaques on the corneas were imaged using a stereomicroscope. Western blot analysis for HSV-1 proteins was performed to verify HSV-1 infection of the cornea. Using the ex vivo model, zinc oxide tetrapods were tested for their anti-HSV-1 potential, and a cytokine profile was developed to assess the effects of the treatment., Results: Cultured corneas and the use of β-galactosidase-expressing HSV-1(KOS)tk12 virus can provide an attractive ex vivo model to visualize and study HSV-1 entry and spread of the infection in tissues. We found that unlike cultured HCE cells, which demonstrated nearly 100% infectivity, HSV-1 infection of the cultured cornea was more restrictive and took longer to develop. We also found that the zinc oxide tetrapod-shaped nano- and microstructures inhibited HSV infection of the cultured cells, as well as the cultured corneas. The cytokine profile of the infected samples was consistent with previous studies of HSV-1 corneal infection., Conclusions: The ability to visualize HSV-1 growth and spread in corneal tissues can provide new details about HSV-1 infection of the cornea and the efficacy of new cornea-specific antiviral drug candidates. The ex vivo model also demonstrates antiviral effects of zinc oxide tetrapods and adequately portrays the drug delivery issues that cornea-specific treatments face.

Purpose: To describe 3 cases of herpes simplex virus (HSV) vesicular blepharitis that progressed to infectious epithelial keratitis despite treatment with oral acyclovir, but responded to topical antiviral therapy., Methods: Retrospective review of a small case series., Results: One adult and 2 children presented with unilateral HSV vesicular blepharitis without evidence of corneal involvement. Each patient was placed on a therapeutic dose of oral acyclovir. While taking oral antiviral therapy, the patients developed HSV infectious epithelial keratitis, which was treated with trifluridine 1% solution 9 times daily in the adult and ganciclovir 0.15% ophthalmic gel 5 times daily in the 2 children. All 3 cases showed resolution of epithelial keratitis within 3 to 10 days after initiation of topical antiviral treatment while oral acyclovir was continued., Conclusions: Oral antiviral therapy alone may not adequately prevent progression of infectious ocular HSV blepharoconjunctivitis. Topical antiviral therapy appeared to enable resolution of HSV epithelial keratitis that arose during oral acyclovir treatment.

Introduction: Primary cultures of human corneal epithelial (HCE) cells usually cease to grow after four or five passages. This result in a small cell yield for experiments such as the eye irritancy test represents a serious problem for human and animal corneal epithelial research. In the present study, we established an HCE cell line immortalized by simian virus 40 (SV40), a polyomavirus, and characterized the inherent morphologic and cytologic cell properties., Methods: Primary cultured HCE cells were infected with a SV40 large T antigen (SV40 T)-expressing retrovirus, and were selected using G418 solution, an aminoglycoside antibiotic. To ensure that the immortalized cell lines express SV40 T and cytokeratin-3, a corneal epithelial-specific marker, we conducted reverse-transcription (RT)-PCR and Western blot analysis., Results: These cell lines continued to grow for more than 50 generations, exhibiting a cobble stone-like appearance similar to normal HCE cells and an increased proliferation rate compared to primary cultured HCE cells. RT-PCR results showed that the immortalized cell lines expressed SV40 T while the primary cultured cells did not. In the Western blot assay, protein levels of phosphorylated (Ser15) p53 protein were significantly decreased in the immortalized cell lines while the expression of total p53 protein was constant. In addition, expression of p21(cip1), a cell cycle protein, was down-regulated in the immortalized cells. Moreover, a cornea epithelium-specific marker, cytokeratin-3 (CK-3), was expressed at equal levels in the immortalized cells and primary HCE cells., Discussion: Taken together, these results indicate that immortalized HCE cell lines were successfully established using the SV40-retroviral vector. These cells may be an excellent model for detecting the adverse effects of standard toxic materials and could replace the traditional eye irritation test as an animal-free alternative method., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: Herpes simplex virus type I (HSV-1) is capable of causing a wide array of human ocular diseases. Herpes simplex virus keratitis (HSK)-induced cytopathogenicity together with the chronic immune-inflammatory reaction can trigger stromal scarring, thinning, and neovascularization which may lead to permanent vision impairment. Lychee flower extract (LFE) is known for its antioxidant and anti-inflammatory effects. Therefore, in this study, we investigated the mechanism of the Statens Seruminstitut rabbit corneal (SIRC) epithelial cells infected by HSV-1 and examined the antiviral capabilities of LFE., Methods: SIRC cells were pretreated with different concentrations of LFE (0.2, 0.1, and 0.05 μg/ml) and then infected with 1 MOI of HSV-1 for 24 h. The cell viability or morphology was evaluated in this study. In addition, the supernatants and cell extracts were collected for Cell Counting Kit-8 (CCK), plaque assay, and western blotting., Results: We found that HSV-1-induced cell proliferation is regulated through inhibition of the mammalian target of rapamycin (mTOR) and p70s6k phosphorylation in response to the LFE. In addition, the LFE enhanced the autophagy protein expression (Beclin-1 and light chain 3, LC3) and decreased the viral titers., Conclusions: These results showed the antiviral capabilities and the protective effects of LFE. Taken together, our data indicate that LFE has potential as an anti-HSK (herpes simplex keratitis) for HSV-1 infection.

Purpose: Aim was to measure the effect of persistent subepithelial corneal infiltrates (SEIs) after epidemic keratoconjunctivitis (EKC) on visual performance and corneal optical quality., Methods: We examined 53 patients divided into two groups. Patients with previous EKC constituted the study group (Group 1, n: 27). Healthy age matched subjects constituted the control group (Group 2, n: 26). Study group was subdivided into eyes with SEI (Group 1A, 40 eyes) and healthy fellow eyes (Group 1B, 14 eyes). Patients were submitted to a complete examination including high- and low-contrast visual acuities (HCVA, LCVA), corneal topography and aberrometry., Results: The mean interval between epidemic conjunctivitis and examination was 7.25 ± 5.63 months. Mean number of corneal infiltrates was 17.30 ± 14.38. The mean HCVA and LCVA were significantly lower in the eyes with SEI than fellow eyes and control group (p = 0.001). LCVA value was also worse in the fellow eyes when compared to control (p = 0.048). Corneal topography values were significantly higher in the eyes with SEI from both fellow eyes and control group. During corneal aberrometry, the mean root mean square value of spheric aberration, irregular astigmatism and total aberration were significantly higher in SEI group when compared to fellow eyes and control group (p < 0.05). Total aberrations were higher than control subjects in the fellow eyes. Trefoil aberration was higher in SEI group only when compared to control subjects, while there was no significant difference in coma aberration values between the groups (p > 0.05)., Conclusion: Results of this study suggested that visual performance is compromised in patients with EKC by aberrations and changes in topographic variables., (© 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Purpose: Aciclovir (ACV), valaciclovir (VACV) and famciclovir (FCV) are used for systemic infections caused by herpes virus. In Japan, only topical ACV is permitted for use against herpetic keratitis. We investigated the effectiveness of topical ACV, oral VACV and oral FCV on mouse epithelial herpetic keratitis., Methods: C57/BL76 mice were inoculated with HSV-1 McKrae strain in the cornea. Once infection was confirmed 4 days after inoculation, topical ACV, oral VACV and FCV were started and administered for 5 days. Control groups were given either topical or oral saline. On days 2, 4, 6 and 10 after medication started, tears, eyeballs, and trigeminal ganglia were examined using viral culture and real-time PCR., Results: Viral culture of tears detected no HSV in the topical ACV group on day 4 after administration start; with similar results for the oral VACV group on day 4; and the oral FCV group on day 6. Real-time PCR of the eyeballs showed significant decrease of HSV DNA copy number in the topical ACV group on days 4 and 6 compared to the topical saline group. Real-time PCR of the trigeminal ganglia showed significant decrease of HSV DNA copy number in the oral VACV group on days 4 and 6, and in the oral FCV group on day 6 compared to the oral saline group., Conclusion: We suggest that 5-day administration of topical ACV, oral VACV and oral FCV are effective for mouse epithelial herpetic keratitis and sufficiently decrease HSV amounts in the ocular surface and eyeballs.

Herpes simplex virus-1 (HSV-1) infection causes severe conditions, with serious complications, including corneal blindness from uncontrolled ocular infections. An important cellular defense mechanism against HSV-1 infection is autophagy. The autophagic response of the host cell was suggested to be regulated by HSV-1. In this study, we performed a detailed analysis of autophagy in multiple HSV-1-targeted cell types, and under various infection conditions that recapitulate a productive infection model. We found that autophagy was slightly inhibited in one cell type, while in other cell types autophagy maintained its basal levels mostly unchanged during productive infection. This study refines the concept of HSV-1-mediated autophagy regulation to imply either inhibition, or prevention of activation, of the innate immune pathway.

Aims: To investigate the role of herpes virus entry mediator (HVEM) in viral entry and inflammatory cytokine production in response to herpes simplex virus (HSV) type 1 challenge in human corneal epithelial cells., Methods: HVEM expression in human corneal epithelial cells was determined by immunofluorescence and flow cytometry. The HSV-1 virus expressing β-galactosidase was used to challenge corneal epithelial cells and viral entry assays were performed to ascertain HSV-1 entry into cells. Levels of cytokines TNF-α, IL-6, IFN-x03B3;, IL-12, and IL-18 and chemokines MIP-1α, MIP-1β and MIP-2 were detected in corneal epithelial cells treated with control or HVEM siRNA in response to HSV-1 challenge., Results: Human corneal epithelial cells were positive for HVEM expression and showed high susceptibility to HSV-1 entry. Silencing of HVEM did not alter viral entry dramatically. However, levels of the cytokine IFN-x03B3; and chemokines MIP-1α and MIP-1β were measured to be higher in HVEM siRNA-treated cells than control after HSV-1 challenge., Conclusions: HVEM in human corneal epithelial cells may act to dampen the production of some cytokines and chemokines and thus it may modulate the innate immune response against HSV-1. This may provide a novel mechanism for the pathogenesis of HSV-1 infection in the cornea., (© 2015 S. Karger AG, Basel.)

Background/aims: Herpes simplex virus (HSV) type I keratitis remains a leading cause of corneal morbidity, despite the availability of effective antiviral drugs. Improved understanding of virus-host interactions at the level of the host DNA damage response (DDR), a known factor in the development of HSV-1 keratitis, may shed light on potential new therapeutic targets. This report examines the role of checkpoint kinase 2 (Chk2), a DDR mediator protein, in corneal epithelial HSV-1 infection., Methods: A small-molecule inhibitor of Chk2 (Chk2 inhibitor II) was applied to HSV-1-infected cultured human corneal epithelial cells (hTCEpi and HCE) as well as to explanted and organotypically cultured human and rabbit corneas. Infection levels were assessed by plaque assay and real-time PCR. RNAi-mediated depletion of Chk2 was performed to confirm the effect of the inhibitor., Results: Inhibition of the Chk2 kinase activity greatly suppresses the cytopathic effect, genome replication and infectious progeny production in vitro and ex vivo., Conclusion: This report demonstrates the critical role of Chk2 kinase in the establishment of HSV-1 corneal epithelial infection. These data contribute to our understanding of herpesvirus-host interactions and underscore the significance of DDR activation in HSV-1 keratitis.

Objective: To determine the safety and efficacy of topical 0.03% tacrolimus ointment treatment for subepithelial corneal infiltrates (SEIs)., Methods: This prospective non-controlled interventional case series included patients with SEIs who had been previously treated with topical corticosteroids with either no improvement or the medication being withdrawn due to associated intraocular pressure (IOP) elevation. The patients were treated with 0.03% tacrolimus ointment twice daily for 22 weeks (including a 1-month washout). The objective data were best-corrected Snellen visual acuity (BCVA), IOP, and full ocular examination results, including SEI severity and the Schirmer test. The subjective data were the patients' responses to a questionnaire at all follow-up visits., Results: The patients consisted of five males (45%) and six females (55%) (mean age 50 ± 11 years) who were followed up for an average of 22 weeks. The mean BCVA (logarithm of the minimum angle of resolution [logMAR]) before and after treatment was 0.34 ± 0.09 and 0.08 ± 0.04 respectively (p = 0.042). All the patients evidenced significant objective clinical improvement, and none had a severe degree of SEI at the end of the treatment. The patients reported considerable reduction in the severity of their symptoms (foreign body sensation, glare, etc.). Three patients were excluded due to side-effects (one had severe dizziness and discomfort), and their data were excluded from the study., Conclusion: Topical tacrolimus 0.03% is a safe and effective alternative treatment in patients with SEIs who do not respond to other treatment modalities or have untoward side-effects from topical steroids.

Purpose: Although Epstein-Barr virus (EBV)-associated keratitis is rare, it can cause acute corneal necrosis and neovascularization. We aimed to examine the signaling mechanism by which EBV causes epithelial-mesenchymal transition (EMT) in human corneal epithelial cells (HCECs) in vitro., Methods: The cellular response to EBV was assessed by real-time PCR, Western blot, migration assay, invasion assay, inhibitor assay, and ELISA assay., Results: A model of EBV-induced EMT was established in HCECs. The EBV induced morphologic changes in the cells; the loss of epithelial markers E-cadherin, ZO-1, and β-catenin; and an increase in the mesenchymal markers N-cadherin, Vimentin, Snail, and TCF8/Zeb1. The EBV infection also led to the nuclear translocation of Snail and TCF8/Zeb1; enhanced the secretion of IL-6, IL-8, VEGF, TGF-β1, TNF-α, and MCP-1; and upregulated the expression of MMP2 and MMP9. The EBV-infected HCECs exhibited increased migration and invasiveness compared to uninfected HCECs. We measured the involvement of Syk, Src, PI3K/Akt, and Erk signaling, but not Smad, in EMT by EBV-induced TGF-β1. We demonstrated that treatment with TGF-β1, TGF-β receptors, Syk, or Src inhibitor blocked TGF-β1, Syk, or Src signaling activation, and EMT development by EBV. Moreover, these inhibitors prevented PI3K/Akt and Erk activation., Conclusions: An EBV infection in HCECs can lead to a mesenchymal fibroblast-like morphology, and cause EMT through the activation of PI3K/Akt and Erk by TGF-β1-mediated Syk and Src signaling. This phenomenon may have implications for EBV-associated keratitis and molecular approaches to treatment.

Purpose: Herpes keratitis (HK) remains the leading cause of cornea-derived blindness in the developed world, despite the availability of effective antiviral drugs. Treatment toxicity and the emergence of drug resistance highlight the need for additional therapeutic approaches. This study examined ataxia telangiectasia mutated (ATM), an apical kinase in the host DNA damage response, as a potential new target for the treatment of HK., Methods: Small molecule inhibitor of ATM (KU-55933) was used to treat herpes simplex virus type 1 (HSV-1) infection in three experimental models: (1) in vitro--cultured human corneal epithelial cells, hTCEpi, (2) ex vivo--organotypically explanted human and rabbit corneas, and (3) in vivo--corneal infection in young C57BL/6J mice. Infection productivity was assayed by plaque assay, real-time PCR, Western blot, and disease scoring., Results: Robust ATM activation was detected in HSV-1-infected human corneal epithelial cells. Inhibition of ATM greatly suppressed viral replication in cultured cells and in explanted human and rabbit corneas, and reduced the severity of stromal keratitis in mice. The antiviral effect of KU-55933 in combination with acyclovir was additive, and KU-55933 suppressed replication of a drug-resistant HSV-1 strain. KU-55933 caused minimal toxicity, as monitored by clonogenic survival assay and fluorescein staining., Conclusions: This study identifies ATM as a potential target for the treatment of HK. ATM inhibition by KU-55933 reduces epithelial infection and stromal disease severity without producing appreciable toxicity. These findings warrant further investigations into the DNA damage response as an area for therapeutic intervention in herpetic ocular diseases.

Purpose: To comparatively analyze the methodological efficacy of the polymerase chain reaction (PCR) assay for herpes simplex virus 1 (HSV) detection in tears., Methods: This retrospective study reviewed the medical records of 115 patients who were clinically diagnosed with herpes keratitis, and their tear samples were collected for HSV detection. PCR positive rates were analyzed for their dependence on the PCR primers used (conventional PCR primer vs. nested PCR primer), the tear collecting method used (micropipetting vs. collection with schirmer strip), the disease manifestation and the patient's previous medication history., Results: HSV DNA was detected in 23 out of 115 (20%) tear samples. The PCR positive rate in tear samples did not differ depending on the PCR primer or tear collection method used. Typical epithelial lesions showed a higher positive rate (31.4%) than atypical epithelial lesions (10.9%). The previous history of the antiviral agent seemed to affect the PCR positive rate., Conclusions: Although the PCR positive rate was not dependent on the tear collection method or primers, HSV detection in tears using PCR was shown to be a supplementary diagnostic test in typical and atypical herpes epithelitis.

Purpose: Epidemic keratoconjunctivitis (EKC) is a severe ocular infection caused by a few types (8, 19a [relabeled as 64 recently], 37, 53, and 54) of human adenoviruses (HAdVs). HAdVs are known for their strong host species specificity that limits studying HAdV virulence and pathophysiology in animal models., Methods: A HAdV infection model of primary porcine corneal epithelial cells (PPCE) and primary porcine corneal keratocytes (PPCK) was established and compared to primary human corneal epithelial cells (PHCE) and primary human corneal keratocytes (PHCK). Induction of interleukin-8 (IL-8) messenger RNA (mRNA), HAdV DNA replication, and the release of infectious virus progeny by the EKC-associated type HAdV-D37 and the non-EKC-associated type HAdV-D22 were studied., Results: PPCE and PPCK morphology and the expression of α2,3-linked sialic acid, the main receptor of EKC-associated HAdV types, were akin to human corneal cells (PHCE and PHCK). Induction of IL-8 mRNA was observed as early as 8 h after HAdV infection. Induction of IL-8 mRNA by HAdV-D37 infection was significantly higher (p≤0.001) than by HAdV-D22 infection in PPCE, PPCK, PHCE, and PHCK. Detection of HAdV-DNA replication, release of infectious virus progeny, and the development of cytopathic effect indicated that PPCE and PPCK were fully permissive for HAdV-D37 and HAdV-D22 replication as were the human corneal cells (PHCE and PHCK). Infectious virus titers after HAdV-D37 infection (1.0 × 10(5) TCID50/ml) were significantly higher (p=0.001) than after HAdV-D22 infection (1.8 × 10(4) TCID50/ml) in PPCE, PHCE, and PHCK but not significantly different in PPCK., Conclusions: Primary porcine epithelial cells and keratocytes are nonhuman corneal cell culture models fully permissive for HAdV infection. The models hold promise for studying the virulence and pathophysiology of EKC-associated adenovirus types compared to other adenovirus types.

Purpose: To investigate the characteristics of the inflammatory response detected in corneas with active non-epithelial herpes simplex virus (HSV) keratitis., Materials and Methods: The study was designed as a cross-sectional study undertaken at a university setting. Twenty-one eyes of 21 patients with a mean age of 40.1 ± 13.0 years (range: 18-70 years) and whose corneal findings were compatible with active HSV keratitis were included. All patients were evaluated with in vivo confocal microscopy (Confoscan 3, Nidek). Fifty healthy, age-matched subjects served as controls. The features and extent of corneal inflammation, degree of involvement of subbasal nerve plexus and endothelial cell density were evaluated., Results: Seven eyes (33.3%) demonstrated stromal keratitis, six (28.6%) endotheliitis and eight (38.1%) presented with keratouveitis. Intraepithelial inflammatory cell infiltration, consisting of dendritic and small round cells, was observed in 11 eyes (52.4%). Stromal involvement was detected in 95.2% of eyes in the form of focal and diffuse cellular infiltrates; of these, anterior, posterior and pan stromal infiltration was observed in 85.7, 71.4 and 47.6% of cases, respectively. No particular keratic precipitate morphology was found to be diagnostic for HSV keratitis. The mean endothelial cell density of eyes with HSV keratitis (2437 ± 599 cells/mm(2)) was significantly lower (-18.8%) than that of controls (3003 ± 250 cells/mm(2)); (p < 0.001). The mean subbasal nerve density of patients with HSV keratitis (474.5 ± 227.8 μm/frame) was significantly lower than that of controls (1589.6 ± 499.0 μm/frame) (p < 0.001)., Conclusions: Non-epithelial HSV keratitis related inflammatory response is characterized by cellular infiltration of all corneal layers, attenuation of the subbasal nerve plexus and endothelial cell loss.

Herpes simplex virus (HSV) is associated with a variety of ocular diseases, including epithelial and stromal keratitis. HSV can cause stromal opacification and is believed to be the leading cause of infectious blindness in the developed world. An improved understanding of the global burden of HSV keratitis, including the incidence of severe vision loss, could have a significant effect on prevention and treatment and place it in perspective among causes of corneal ulceration. We found that the global incidence of HSV keratitis is roughly 1.5 million, including 40,000 new cases of severe monocular visual impairment or blindness each year. We also discuss relevant epidemiologic issues regarding HSV epithelial and stromal disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: To evaluate the treatment with topical 0.05% cyclosporine A (CsA) in patients with subepithelial corneal infiltrates (SEI)., Methods: We reviewed 16 patients (22 eyes) before and after the treatment with 0.05% CsA eye drops. All patients had been treated previously with topical corticosteroids without any improvement and also they had to stop the medication secondary to intraocular pressure elevation. The objective data recorded included best-corrected visual acuity (BCVA), evaluation of corneal subepithelial infiltrate scores (CSIS), intraocular pressure (IOP) prior to treatment and the last follow-up visit., Results: Six males (37.5%) and 10 females (62.5%), mean age of 35.2 ± 16.6 years, were included. The patients' average topical CsA use duration was 5.1 ± 3.5 months (1 - 13 months). The average follow up time of the patients was 9.2 ± 4.7 months (4 - 22 months). One patient, although he didn't have a 0 scale of SCIS, did not show up for follow up examinations after six months. The mean BCVA (logarithm of the minimum angle of resolution) before and after the treatment were 0.15 ± 0.15 and 0.07 ± 0.07 respectively, CSIS 1.68 ± 0.89 and 0.23 ± 0.53 respectively, IOP 18.50 ± 3.82 and 16.86 ± 2.76 mmHg respectively. There were statistically significant improvements in BCVA (p=0.002), reduction of CSIS (p=0.002) and reduction of IOP (p<0.001) prior to treatment and the last follow-up visit. 18 eyes (81.9%) showed clinical improvement and 4 (18.1%) had decreased SEI which did not fully disappear during the treatment period. The eyes which reached CSIS score 0 (18 eyes) were treated with CsA for 1 - 13 months; while the eyes which had clinical improvement but had not CSIS score 0 (4 eyes) were decided to discontinue of CsA treatment in last follow-up visit. There were recurrences in 2 eyes 3 months after the treatment. Patients reported reduction in the severity of symptoms after the treatment. Most of the patients reported no foreign body sensation, glare, or other side effects with topical CsA treatment. Overall, patients noted an improvement in vision and satisfaction with topical 0.05% CsA treatment., Conclusions: Topical 0.05% CsA is a safe and effective alternative treatment in patients with SEI who do not respond to other treatment modalities or have undesired side effects from topical steroids.

Purpose: To report the use of photorefractive keratectomy (PRK) with mitomycin-C (MMC) to treat concomitant myopia and visually significant infiltrates associated with epidemic keratoconjunctivitis (EKC)., Setting: Ophthalmic Consultants of Long Island, Nassau University, Long Island, New York, USA., Design: Interventional case series., Methods: Consecutive patients with myopia and recalcitrant subepithelial infiltrates after EKC were treated with custom wavefront PRK (Visx S4 IR) and MMC with a target of emmetropia in all cases., Results: The study evaluated 6 eyes of 3 patients. One year after treatment, all eyes attained an uncorrected distance visual acuity of 20/20 or better. There was no recurrence of infiltrates within the ablation zone in any eye., Conclusion: The use of topical MMC in conjunction with PRK to treat subepithelial infiltrates due to EKC provided good visual and refractive results., (Copyright © 2012 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Toll-like receptors (TLRs) are innate sentinels required for clearance of bacterial and fungal infections of the cornea, but their role in viral immunity is currently unknown. We report that TLR signaling is expendable in herpes simplex virus (HSV)-1 containment as depicted by plaque assays of knockout mice (MyD88(-/-), Trif(-/-) and MyD88(-/-) Trif(-/-) double knockout) resembling wild-type controls. To identify the key sentinel in viral recognition of the cornea, in vivo knockdown of the DNA sensor IFI-16/p204 in the corneal epithelium was performed and resulted in a loss of IFN-regulatory factor-3 (IRF-3) nuclear translocation, interferon-α production, and viral containment. The sensor seems to have a similar function in other HSV clinically relevant sites such as the vaginal mucosa in which a loss of p204/IFI-16 results in significantly more HSV-2 shedding. Thus, we have identified an IRF-3-dependent, IRF-7- and TLR-independent innate sensor responsible for HSV containment at the site of acute infection.

Purpose: Primary and recurrent infections of the cornea by herpes simplex virus 1 (HSV-1) are important causes of eye disease. Three unrelated classes of glycoprotein D receptors for HSV-1 entry into cells have been identified. This study was undertaken to uncover the relative significance of nectin-1 as an entry receptor in corneal infection and HSV-1 spread to the trigeminal ganglia (TG), a site important for HSV-1 latency and recurrent corneal infection., Methods: To assess the significance of nectin-1, a member of the immunoglobulin superfamily, in primary HSV-1 infection and spread to the TG, we used a murine model of corneal infection and a HSV-1 mutant, KOS(Rid1), which can only use nectin-1 for entry. Immunohistochemistry, real-time PCR, and plaque assays using HSV-1 infected tissues were performed., Results: We demonstrated that receptor usage by HSV-1 limited to nectin-1 does not significantly change the spread of HSV-1 in the corneal epithelium during primary infection. We also found that nectin-1-specific entry does not affect the capacity of the virus to spread to the TG from the cornea., Conclusions: Our findings suggest that nectin-1 alone is sufficient for HSV-1 entry into the cornea and spread to the TG.

Aim: Study of innate immunity genes expression (TLR9 and BD-2) in epithelial cells of mice cornea on the model of viral keratitis during the application of immunotherapy., Materials and Methods: RNA isolation from cornea cells was performed by using RNeasy Mini Kit (Qiagen) and "RIBO-sorb" (ILS, Russia) reagent kits. "Kit for performing PCR-RT in the presence of intercalating dye SYBR Green I" (Syntol, Russia) was used for performing PCR. Data on gene expression is presented in decimal logarithms (relatively to 1 million of beta-actin gene copies)., Results: In C57B1/6 mice line at day 1 post infection the virus was detected in 33% of animals, at 3 days - in 90%, at 7 days the number of infected mice started to decline. In cornea of the mice infected with HSV-1 a significant decrease of TLR9 gene expression was observed. Administration of a complex of natural cytokines and antimicrobial peptides (Superlimf preparation) caused statistically significant increase of expression levels of the studied TLR9 and BD-2 genes., Conclusion: The model of viral keratitis and expression analysis of recognizing receptor TLR9 and defensin (BD-2) genes can be used to study mechanism of action of various preparations during infection in eye tissues and evaluation of possibility of their use in complex therapy of ophthalmic herpes.

Purpose: To describe the use of cyclosporine A (CSA) 1% eye drops for the treatment of symptomatic corneal subepithelial infiltrates (SEI) occurring as a sequelae of adenoviral keratoconjunctivitis (AK) that are resistant to tapering of corticosteroid eye drops., Methods: This is a retrospective case series of patients seen at 2 institutions who had symptomatic corneal SEI occurring after AK that was resistant to tapering of corticosteroid eye drops and who were subsequently treated with CSA 1%. Information gathered included basic demographic information (age and sex), involved eye(s), duration of symptoms, initial best spectacle-corrected visual acuity (BSCVA), type of corticosteroid used, clinical course, and best spectacle-corrected visual acuity at the last follow-up visit., Results: Twelve eyes of 7 patients had symptomatic SEI develop after AK that were responsive to corticosteroid eye drops but were resistant to tapering. After the initiation of CSA eye drops, the corticosteroid eye drops could be tapered, and all eyes could be maintained on CSA eye drops once per day or less. Mean follow-up time was 13.0 months (range, 4-28 months)., Conclusions: CSA eye drops may be an effective corticosteroid-sparing agent for the treatment of SEI after AK. The use of CSA in this setting warrants further study.

Purpose: To determine the roles played by toll-like receptor 9 (TLR9) in cultured human corneal endothelial (HCEn) cells after herpes simplex virus type 1 (HSV-1) infection and to characterize the TLR9-mediated antiviral responses., Method: Immortalized HCEn cells were examined for TLR expression. The upregulation of inflammatory cytokines after HSV-1 infection was determined by real-time RT-PCR or protein array analyses. The TLR9-mediated HSV-1 replication was determined by real-time PCR and plaque assay. To determine whether there was an activation of the signal transduction pathway, HCEn cells that were transfected with pathway-focused transcription factor reporters were examined for promoter activity., Results: TLR9 was abundantly expressed intracellularly in HCEn cells. The CpG oligonucleotide, a TLR9 ligand, stimulated the NF-κB activity in HCEn cells. HSV-1 infection also stimulated NF-κB and induced NF-κB -related inflammatory cytokines, including RANTES, IP-10, MCP-2, MIF, MCP-4, MDC, MIP-3α, IL-5, TARC, MCP-1, and IL-6. The induction of these cytokines was significantly reduced by blocking the activity of TLR9. In addition, viral replication in HCEn cells was significantly reduced by the inhibition of TLR9, but was preserved by a concomitant activation of the NF-κB cascade. Of the different HSV-1-induced inflammatory cascade-related transcription factors, TLR9 was found to activate NF-κB, cyclic AMP response element (CRE), and the CCAAT-enhancer-binding proteins (C/EBP) the most., Conclusions: Corneal endothelial cells transcriptionally initiate inflammatory programs in response to HSV-1 infection related to NF-κB, CRE, and C/EBP and express arrays of inflammatory cytokine induction by TLR9. On the other hand, HSV-1 exploits TLR9-mediated NF-κB activation for its own replication.

Adenovirus type 37 (Ad37) is a leading cause of epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular disease. Whereas most other adenoviruses infect cells by engaging CD46 or the coxsackie and adenovirus receptor (CAR), Ad37 binds previously unknown sialic acid-containing cell surface molecules. By glycan array screening, we show here that the receptor-recognizing knob domain of the Ad37 fiber protein specifically binds a branched hexasaccharide that is present in the GD1a ganglioside and that features two terminal sialic acids. Soluble GD1a glycan and GD1a-binding antibodies efficiently prevented Ad37 virions from binding and infecting corneal cells. Unexpectedly, the receptor is constituted by one or more glycoproteins containing the GD1a glycan motif rather than the ganglioside itself, as shown by binding, infection and flow cytometry experiments. Molecular modeling, nuclear magnetic resonance and X-ray crystallography reveal that the two terminal sialic acids dock into two of three previously established sialic acid-binding sites in the trimeric Ad37 knob. Surface plasmon resonance analysis shows that the knob-GD1a glycan interaction has high affinity. Our findings therefore form a basis for the design and development of sialic acid-containing antiviral drugs for topical treatment of EKC.

Herpes simplex virus type-1 (HSV-1) is a common human pathogen that relies heavily on cell-to-cell spread for establishing a lifelong latent infection. Molecular aspects of HSV-1 entry into host cells have been well studied; however, the molecular details of the spread of the virus from cell-to-cell remain poorly understood. In the past, the role of heparan sulfate proteoglycans (HSPG) during HSV-1 infection has focused solely on the role of HS chains as an attachment receptor for the virus, while the core protein has been assumed to perform a passive role of only carrying the HS chains. Likewise, very little is known about the involvement of any specific HSPGs in HSV-1 lifecycle. Here we demonstrate that a HSPG, syndecan-1, plays an important role in HSV-1 induced membrane fusion and cell-to-cell spread. Interestingly, the functions of syndecan-1 in fusion and spread are independent of the presence of HS on the core protein. Using a mutant CHO-K1 cell line that lacks all glycosaminoglycans (GAGs) on its surface (CHO-745) we demonstrate that the core protein of syndecan-1 possesses the ability to modulate membrane fusion and viral spread. Altogether, we identify a new role for syndecan-1 in HSV-1 pathogenesis and demonstrate HS-independent functions of its core protein in viral spread.

Unlabelled: Abstract Purpose: To determine if a peptide, TAT-Cd(0), inhibits Herpes simplex virus type 1 infection of human corneal epithelial cells., Methods: TAT-Cd(0) and a control peptide, E(50,51)TAT-Cd(0), were added at various times throughout infection with the lacz-expressing hrR3 virus, and viral replication was measured by β-galactosidase activity. Toxicity was assessed using a dye reduction assay., Results: The CC(50) value for TAT-Cd(0) was ∼100 μM. In assays with peptide present at all times, TAT-Cd(0) was 150-fold more active than E(50,51)TAT-Cd(0) (EC(50) 0.2 vs. 30.0 μM). The EC(50) values of TAT-Cd(0) for entry inhibition, cell protection, virus inactivation, and inhibition of attachment were 0.1, 0.4, 9.5, and 3.0 μM, respectively. TAT-Cd(0) was less effective when added 1 h postinfection (EC(50) = 30.0 μM)., Conclusions: TAT-Cd(0) is an effective inhibitor of Herpes simplex virus type 1 infection in human corneal epithelial cells and affects multiple steps before, or very early, in infection. The peptide has potential as an antiviral and further studies are warranted.