Rapid NETosis Is an Effector Mechanism to Combat Ocular Herpes Infection.

Purpose: Neutrophils are known mediators of innate immunity, yet their effector function in herpesvirus infections remains poorly understood. Here, we elucidate the mechanistic action and pivotal role of neutrophil extracellular traps (NETs) during herpes simplex virus type 1 (HSV-1) ocular infection. Methods: Neutrophils were collected from mice for HSV-1 infection, fluorescence imaging, and immunoblotting assay. Tear samples from healthy subjects and patients with HSV-1 and mice were collected at L. V. Prasad Eye Institute, India, and at the University of Illinois, USA, respectively. For the in vivo study, C57BL/6 mice as well as diversity outbred mice were infected with HSV-1 (McKrae strain) followed by tear fluid collection at various time points (0-10 days). Samples were used for Flow cytometry, ELISA, and immunofluorescence assay. Human transcriptomic profile of keratitis dataset was used evaluate NETosis signaling pathways. We also performed neutrophil depletion studies. Results: Our data revealed a discernible temporal NET formation (NETosis) predominantly in the infected eye, across normal and diversity outbred murine models and human cases of HSV-1 infection. HSV-1 instigates swift NETosis governed by caspase-1 activation and myeloperoxidase secretion. Distinct accumulations of neutrophils, remaining unengaged in NET release in the contralateral eye post-infection, hinting at a proactive defensive posture in the uninfected eye. Moreover, neutrophil depletion accentuated ocular pathology, augmented viral load, and escalated disease scores, substantiating the protective effects of NETs in curtailing viral replication. Conclusions: Our report uncovers a previously unexplored mechanism of NETosis through pro-inflammatory cell death in response to ocular HSV-1 infection, and HPSE up-regulation, identifying new avenues for future studies.

Transcriptome Analysis of Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Our previous studies have shown that systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spread to the eye with subsequent establishment of latency in choroid/RPE. In this study, RNA sequencing (RNA-Seq) analysis was used to determine the molecular genetic changes and pathways affected by ocular MCMV latency. MCMV (50 pfu per mouse) or medium as control were injected intra-peritoneally (i.p.) into BALB/c mice at <3 days after birth. At 18 months post injection, the mice were euthanized, and the eyes were collected and prepared for RNA-Seq. Compared to three uninfected control eyes, we identified 321 differentially expressed genes (DEGs) in six infected eyes. Using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA), we identified 17 affected canonical pathways, 10 of which function in neuroretinal signaling, with the majority of DEGs being downregulated, while 7 pathways function in upregulated immune/inflammatory responses. Retinal and epithelial cell death pathways involving both apoptosis and necroptosis were also activated. MCMV ocular latency is associated with upregulation of immune and inflammatory responses and downregulation of multiple neuroretinal signaling pathways. Cell death signaling pathways are also activated and contribute to the degeneration of photoreceptors, RPE, and choroidal capillaries.

Molecular imaging on ACE2-dependent transocular infection of coronavirus.

A transocular infection has been proved as one of the main approaches that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the body, and angiotensin-converting enzyme 2 (ACE2) plays a key role in this procedure. Dynamic and quantitative details on virus distribution are lacking for virus prevention and drug design. In this study, a radiotraceable pseudovirus packed with an enhanced green fluorescent protein (EGFP) gene, 125 I-CoV, was prepared and inoculated in the unilateral eye of humanized ACE2 (hACE2) mice or ACE2-knockout (ACE2-KO) mice. Single-photon emission computed tomography/computed tomography images were acquired at multiple time points to exhibit ACE2-dependent procedures from invasion to clearance. Positron emission tomography (PET) and western blot were performed to quantify ACE2 expression and verify the factors affecting transocular infection. For the transocular infection of coronavirus (CoV), the renin-angiotensin-aldosterone system (RAAS), lungs, intestines, and genital glands were the main targeted organs. Due to the specific anchor to ACE2-expressed host cells, virus concentrations in genital glands, liver, and lungs ranked the top three most and stabilized at 3.75 ± 0.55, 3.30 ± 0.25, and 2.10 ± 0.55% inoculated dose (ID)/mL at 48 h post treatment. Meanwhile, ACE2-KO mice had already completed the in vivo clearance. In consideration of organ volumes, lungs (14.50 ± 3.75%ID) and liver (10.94 ± 0.71%ID) were the main in-store reservoirs of CoV. However, the inoculated eye (5.52 ± 1.85%ID for hACE2, 5.24 ± 1.45%ID for ACE2-KO, p > 0.05) and the adjacent brain exhibited ACE2-independent virus infection at the end of 72 h observation, and absolute amount of virus played a key role in host cell infection. These observations on CoV infection were further manifested by infection-driven intracellular EGFP expression. ACE2 PET revealed an infection-related systematic upregulation of ACE2 expression in the organs involved in RAAS (e.g., brain, lung, heart, liver, and kidney) and the organ that was of own local renin-angiotensin system (e.g., eye). Transocular infection of CoV is ACE2-dependent and constitutes the cause of disturbed ACE2 expression in the host. The brain, genital glands, and intestines were of the highest unit uptake, potentially accounting for the sequelae. Lungs and liver were of the highest absolute amount, closely related to the respiratory diffusion and in vivo duplication. ACE2 expression was upregulated in the short term after infection with CoV. These visual and quantitative results are helpful to fully understanding the transocular path of SARS-CoV-2 and other CoVs.

The Presence of SARS-CoV-2 in Conjunctival Secretions of COVID-19 Patients.

Purpose: To evaluate the presence of SARS-CoV-2 in conjunctival secretions of COVID-19 patients.Material and Methods: In this retrospective study, the records were examined of patients who were treated in the hospital with the diagnosis of COVID-19 between March-May 2020 and were referred to the eye clinic due to ocular symptoms. Conjunctival swabs from both confirmed and suspected COVID-19 cases during hospitalization were analyzed.Results: A total of 35 patients (22 suspected, 13 laboratory-confirmed COVID-19) were referred to the eye clinic. Conjunctival swab samples from 3 patients yielded positive PCR results. These three patients were being treated in the intensive care unit, and all were suspected COVID-19 patients.Conclusion: SARS-CoV-2 may be detected in patients with suspected COVID-19. Even with conjunctivitis findings, SARS-CoV-2 may not be detected in most conjunctiva swab samples of COVID-19 patients.

IL-1α Modulates IFN-γ-Induced Production of CXCL9/MIG during Herpes Simplex Virus Type-1 Corneal Infection.

PURPOSE: Investigating the modulation of neutrophil production of MIG and IP-10 during the inflammatory response to HSV-1 infection. MATERIALS AND METHODS: An ex vivo model of human corneal infection by HSV-1 was used for this study. This model permits the study of cytokine production by human corneal buttons in the presence, or absence, of gradient purified human neutrophils, under conditions of HSV-1 infection. All experimental samples were stimulated with a baseline concentration of recombinant human IFN-γ at 1 ng/mL. The relative levels of production for 12 pro-inflammatory mediators were screened using a multi-analyte ELISA assay. Neutrophil production of chemokines MIG and IP-10, under conditions of IFN-γ and/or HSV-1 stimulation were measured by quantitative ELISA. Lastly, antibody neutralization (goat IgG anti-human IL-1α, 2 µg/mL) of de novo production of IL-1α by corneal tissue was performed to investigated the effect on MIG and IP-10 production in the ex vivo model for HSV-1 infection. RESULTS: Four of the 12 pro-inflammatory mediators screened (IL-8, IL-6, IL-1α and IL-1ß) demonstrated elevated levels of production during corneal cell infection with HSV-1 and communication with neutrophils. Neutrophils were demonstrated to produce significant levels of both MIG and IP-10 under conditions of IFN-γ stimulation, and production of MIG was further upregulated by co-stimulation with IFN-γ and HSV-1. Neutralization of de novo IL-1α production in the model resulted in increased production of the chemokine production MIG but had no observable effect on IP-10 production. CONCLUSIONS: Our data provide evidence demonstrating the potential for expression patterns of MIG and IP-10 to be modulated by IL-1α, during the inflammatory response to HSV-1 corneal infection. Both corneal cells and neutrophils contribute to the production of T cell recruiting chemokines. However, IL-1α has the potential to upregulate MIG production by corneal cells while down-regulating MIG production by neutrophils.

Effects of ROCK Inhibitors on Apoptosis of Corneal Endothelial Cells in CMV-Positive Posner-Schlossman Syndrome Patients.

Purpose: To examine the role of aqueous tumor necrosis factor α (TNF-α)-RhoA-Rho kinase (ROCK) signaling in cytomegalovirus (CMV)-induced apoptosis and the barrier function of cultured human corneal endothelial cells (hCECs) in CMV-positive Posner-Schlossman syndrome (CMV+/PSS) patients. Methods: Aqueous levels of TNF-α, IL-8, IL-10, and several other cytokines in 19 CMV+/PSS patients and 20 healthy control subjects were quantitated using a multiplex assay. The expression of active RhoA in hCECs post-CMV infection was determined using western blotting (WB). The expression levels of TNF-α and nuclear factor kappa B (NF-κB) in CMV-infected hCECs were examined by immunocytochemistry (ICC) and WB with and without ROCK inhibitors. The apoptotic rate and barrier integrity in CMV-infected hCECs were also examined. Results: The expression levels of TNF-α, monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-10 were upregulated in the aqueous humor of CMV+/PSS patients, and among these upregulated cytokines aqueous TNF-α was negatively correlated with the number of corneal endothelial cells. In CMV-infected hCECs, upregulation of TNF-α and NF-κB was determined by WB and ICC. In hCECs, CMV infection induced apoptosis and significantly impaired cell-cell contacts, effects that were attenuated by treatment with a ROCK inhibitor. Conclusions: Aqueous TNF-α was upregulated in CMV+/PSS patients, which may have triggered corneal endothelial cell loss. Modulation of TNF-α, including its downstream Rho-ROCK signaling, could serve as a novel treatment modality for corneal endothelial cell loss in CMV+/PSS patients.

HSV-1 Hijacks the Host DNA Damage Response in Corneal Epithelial Cells through ICP4-Mediated Activation of ATM.

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.

ACE2 and TMPRSS2 are expressed on the human ocular surface, suggesting susceptibility to SARS-CoV-2 infection.

PURPOSE: Conjunctival signs and symptoms are observed in a subset of patients with COVID-19, and SARS-CoV-2 has been detected in tears, raising concerns regarding the eye both as a portal of entry and carrier of the virus. The purpose of this study was to determine whether ocular surface cells possess the key factors required for cellular susceptibility to SARS-CoV-2 entry/infection. METHODS: We analyzed human post-mortem eyes as well as surgical specimens for the expression of ACE2 (the receptor for SARS-CoV-2) and TMPRSS2, a cell surface-associated protease that facilitates viral entry following binding of the viral spike protein to ACE2. RESULTS: Across all eye specimens, immunohistochemical analysis revealed expression of ACE2 in the conjunctiva, limbus, and cornea, with especially prominent staining in the superficial conjunctival and corneal epithelial surface. Surgical conjunctival specimens also showed expression of ACE2 in the conjunctival epithelium, especially prominent in the superficial epithelium, as well as weak or focal expression in the substantia propria. All eye and conjunctival specimens also expressed TMPRSS2. Finally, Western blot analysis of protein lysates from human corneal epithelium obtained during refractive surgery confirmed expression of ACE2 and TMPRSS2. CONCLUSIONS: Together, these results suggest that ocular surface cells including conjunctiva are susceptible to infection by SARS-CoV-2, and could therefore serve as a portal of entry as well as a reservoir for person-to-person transmission of this virus. This highlights the importance of safety practices including face masks and ocular contact precautions in preventing the spread of COVID-19 disease.

Herpes Simplex DNA in Tears of Atypical Dendritic Keratitis and Multiple Punctate Subepithelial Stromal Opacity: A Case Report.

PURPOSE: To report an atypical presentation of herpes simplex virus (HSV) keratitis followed up using expression levels of HSV DNA in tears. METHODS: A 22-year-old Japanese woman with hyperemia and foreign body sensation in her left eye was diagnosed with atypical dendritic keratitis. A slit-lamp examination at presentation indicated the presence of a rush of dendritic lesions with a sparse branching pattern and poor development of terminal bulbs; follicular conjunctivitis was also observed. Positivity for house-dust-mite- and cedar pollen-specific IgE antibodies in her serum indicated atopic diathesis. The HSV DNA levels in her tears were measured by a real-time polymerase chain reaction. RESULTS: At the initial visit, the HSV DNA levels in tears were 6.4 × 10 copies/sample in the right eye and 1.6 × 10 copies/sample in the left eye. The keratitis improved after treatment with topical acyclovir ointment, 5 times a day for 7 days, and systemic valacyclovir 1000 mg/d for 5 days. Multiple punctate subepithelial opacities developed in her left eye on day 7, with undetectable HSV DNA in tears, bilaterally. CONCLUSIONS: We have successfully monitored the HSV DNA levels in tears using quantitative real-time polymerase chain reaction in HSV keratitis where the corneal findings progressed from atypical dendritic keratitis to multiple punctate corneal subepithelial opacities during the treatment period.

Blockade of IL-27 signaling ameliorates herpes stromal keratitis with upregulated CD4+ Foxp3+ regulatory T cells influx in mice.

Purpose: The purpose of this study was to investigate the production of IL-27 p28 and EBI3 in the ocular inflammatory sites, and the role of IL-27 signaling in a model of HSV-1 induced herpetic stromal keratitis (HSK). Methods: The BALB/c mice were injected intraperitoneally (24 h before infection) with anti-IL-27 antibody or IgG antibody as control, infected with HSV-1 via corneal scarification, and then injected intraperitoneally with anti-IL-27 antibody or IgG antibody at 1, 3, and 5 days postinfection. Slit lamp and histopathology were used to assess disease outcome. The levels of IL-27 p28 and EBI3 in corneas were determined by western blotting and immunofluorescence. Furthermore, viral titers were determined, and immune cell infiltrates were collected and analyzed by flow cytometry. Results: We found that the levels of IL-27 p28 and EBI3 in corneas were elevated significantly at the peak of HSK, and both of them were expressed simultaneously in the epithelium, stroma, and endothelium of corneas. In the group of anti-IL-27 treatment, the severity of the corneal lesion and CD4+ T cells infiltration were significantly decreased, and the percentage of CD4+ Foxp3+ Tregs was upregulated markedly in the spleen, DLNs and cornea of HSK mice compared to IgG treatment. Conclusion: These results provided evidence that IL-27 as a pathogenic pro-inflammatory cytokine controlled CD4+ Foxp3+ Tregs production in HSK, which ultimately resulted in promoting the progression of HSK and poor prognosis.

Stability, safety, and pharmacokinetics of ganciclovir eye drops prepared from ganciclovir for intravenous infusion.

PURPOSE: Hospital-prepared topical ganciclovir eye drops made from intravenous infusions are used to treat cytomegalovirus corneal endotheliitis. This study assessed the efficacy of these eye drops. STUDY DESIGN: Experimental study design. METHODS: Ganciclovir solutions (0.5% and 1.0%) prepared by diluting DENOSINE® IV Infusion in saline were stored light-shielded at 4, 25, or 37°C for 12 weeks. Every two weeks during storage, macroscopic evaluation was conducted and ganciclovir concentrations were determined by high performance liquid chromatography. Ocular surface toxicity and corneal ganciclovir concentrations were evaluated following topical instillation of ganciclovir solutions in rabbits. RESULTS: Ganciclovir solutions maintained transparency for 6 weeks, with precipitation appearing after 8 weeks. Ganciclovir concentrations were maintained at ~100% for 6 weeks at 4°C and 25°C and decreased gradually to 90% after 12 weeks. At 37°C, ganciclovir concentrations decreased linearly for 12 weeks. Rabbit eyes showed no ocular surface toxicity. Following instillation of 0.5% ganciclovir solution, endothelial ganciclovir concentrations were 28.0 µg/g at one hour and 4.3 µg/g at three hours. CONCLUSIONS: Ganciclovir eye drops seem to be safe and penetrate the corneal endothelium. The drug in eye drop form is chemically stable for up to 6 weeks. Eye drops' development for approval by regulatory authorities, especially with improved long-term stability, is anticipated.

The Latency-Associated Transcript Inhibits Apoptosis via Downregulation of Components of the Type I Interferon Pathway during Latent Herpes Simplex Virus 1 Ocular Infection.

The herpes simplex virus (HSV-1) latency-associated transcript (LAT) has been shown to inhibit apoptosis via inhibiting activation of proapoptotic caspases. However, the mechanism of LAT control of apoptosis is unclear, because LAT is not known to encode a functional protein, and the LAT transcript is found largely in the nucleus. We hypothesized that LAT inhibits apoptosis by regulating expression of genes that control apoptosis. Consequently, we sought to establish the molecular mechanism of antiapoptosis functions of LAT at a transcriptional level during latent HSV-1 ocular infection in mice. Our results suggest the following. (i) LAT likely inhibits apoptosis via upregulation of several components of the type I interferon (IFN) pathway. (ii) LAT does not inhibit apoptosis via the caspase cascade at a transcriptional level or via downregulating Toll-like receptors (TLRs). (iii) The mechanism of LAT antiapoptotic effect is distinct from that of the baculovirus inhibitor of apoptosis (cpIAP) because replacement of LAT with the cpIAP gene resulted in a different gene expression pattern than in either LAT+ or LAT- viruses. (iv) Replacement of LAT with the cpIAP gene does not cause upregulation of CD8 or markers of T cell exhaustion despite their having similar levels of latency, further supporting that LAT and cpIAP function via distinct mechanisms.IMPORTANCE The HSV-1 latency reactivation cycle is the cause of significant human pathology. The HSV-1 latency-associated transcript (LAT) functions by regulating latency and reactivation, in part by inhibiting apoptosis. However, the mechanism of this process is unknown. Here we show that LAT likely controls apoptosis via downregulation of several components in the JAK-STAT pathway. Furthermore, we provide evidence that immune exhaustion is not caused by the antiapoptotic activity of the LAT.

An Intraocular Inflammatory Profile of Rubella Associated Uveitis.

Purpose: To analyze intraocular cytokine levels and cell profiles in patients with rubella virus-associated uveitis (RVU). Methods: We collected intraocular fluid samples from patients with RVU (n = 10), uveitis of other causes (n = 27), and cataract (n = 22). Levels of 15 cytokines (IL-1ß, IL-1ra, IL-2, IL-6, IL-6rα, IL-7, IL-8, IL-10, IL-17A, IL-23, TARC, MCP-1, TNF-α, PlGF, and VEGF) were measured using multiplex assay, and intraocular cell populations were determined by multiparameter flowcytometry. Clinical characteristics of RVU patients were collected and compared to laboratory outcomes. Results: RVU patients exhibited high intraocular levels of MCP-1, IL-6rα, and TARC, whilst patients with noninfectious uveitis were characterized by high levels of PlGF. Cataract patients showed high levels of IL-2 and IL-23. Intraocular cell population of RVU patients disclosed mainly T-cells and monocytes/macrophages and B-cells were scarcely detected. Conclusion: RVU patients exhibit a cytokine profile distinct from noninfectious uveitis and cataract.

Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo.

Herpes Simplex Virus type 2 (HSV-2) is a neurotropic human pathogen. Upon de novo infection, the viral infected cell protein 0 (ICP0) is immediately expressed and interacts with various cellular components during the viral replication cycle. ICP0 is a multifunctional regulatory protein that has been shown to be important for both efficient viral replication and virus reactivation from latency. In particular, as previously demonstrated in transfected tissue culture models, ICP0 interacts with the cellular E3 ubiquitin ligase SIAH-1, which targets ICP0 for proteasomal degradation. However, the consequence of this virus-host interaction during the establishment of HSV-2 infection in vivo has not yet been elucidated. Here we confirmed that ICP0 of HSV-2 interacts with SIAH-1 via two conserved PxAxVxP amino acid binding motifs. We also demonstrate in vitro that a SIAH-1 binding-deficient HSV-2 strain, constructed by homologous recombination technology, exhibits an attenuated growth curve and impaired DNA and protein synthesis. This attenuated phenotype was also confirmed in an in vivo ocular infection mouse model. Specifically, viral load of the SIAH-1 binding-deficient HSV-2 mutant was significantly reduced in the trigeminal ganglia and brain stem at day 5 and 7 post infection. Our findings indicate that the interplay between ICP0 and SIAH-1 is important for efficient HSV-2 replication in vivo, thereby affecting viral dissemination kinetics in newly infected organisms, and possibly revealing novel targets for antiviral therapy.

Transcriptional Profile and Integrative Analyses of Long Noncoding RNAs in Primary Human Corneal Epithelial Cells in Response to HSV-1 Infection.

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.

Depletion of the Receptor-Interacting Protein Kinase 3 (RIP3) Decreases Photoreceptor Cell Death During the Early Stages of Ocular Murine Cytomegalovirus Infection.

Purpose: The purpose of this study was to determine if the receptor-interacting protein kinase 3 (RIP3) plays a significant role in innate immune responses and death of bystander retinal neurons during murine cytomegalovirus (MCMV) retinal infection, by comparing the innate immune response and cell death in RIP3-depleted mice (Rip3-/-) and Rip3+/+ control mice. Methods: Rip3-/- and Rip3+/+ mice were immunosuppressed (IS) and inoculated with MCMV via the supraciliary route. Virus-injected and mock-injected control eyes were removed at days 4, 7, and 10 post infection (p.i.) and markers of innate immunity and cell death were analyzed. Results: Compared to Rip3+/+ mice, significantly more MCMV was recovered and more MCMV-infected RPE cells were observed in injected eyes of Rip3-/- mice at days 4 and 7 p.i. In contrast, fewer TUNEL-stained photoreceptors were observed in Rip3-/- eyes than in Rip3+/+ eyes at these times. Electron microscopy showed that significantly more apoptotic photoreceptor cells were present in Rip3+/+ mice than in Rip3-/- mice. Immunohistochemistry showed that the majority of TUNEL-stained photoreceptors died via mitochondrial flavoprotein apoptosis-inducing factor (AIF)-mediated, caspase 3-independent apoptosis. The majority of RIP3-expressing cells in infected eyes were RPE cells, microglia/macrophages, and glia, whereas retinal neurons contained much lower amounts of RIP3. Western blots showed significantly higher levels of activated nuclear factor-κB and caspase 1 were present in Rip3+/+ eyes compared to Rip3-/- eyes. Conclusions: Our results suggest that RIP3 enhances innate immune responses against ocular MCMV infection via activation of the inflammasome and nuclear factor-κB, which also leads to inflammation and death of bystander cells by multiple pathways including apoptosis and necroptosis.

Influence of an immunodominant herpes simplex virus type 1 CD8+ T cell epitope on the target hierarchy and function of subdominant CD8+ T cells.

Herpes simplex virus type 1 (HSV-1) latency in sensory ganglia such as trigeminal ganglia (TG) is associated with a persistent immune infiltrate that includes effector memory CD8+ T cells that can influence HSV-1 reactivation. In C57BL/6 mice, HSV-1 induces a highly skewed CD8+ T cell repertoire, in which half of CD8+ T cells (gB-CD8s) recognize a single epitope on glycoprotein B (gB498-505), while the remainder (non-gB-CD8s) recognize, in varying proportions, 19 subdominant epitopes on 12 viral proteins. The gB-CD8s remain functional in TG throughout latency, while non-gB-CD8s exhibit varying degrees of functional compromise. To understand how dominance hierarchies relate to CD8+ T cell function during latency, we characterized the TG-associated CD8+ T cells following corneal infection with a recombinant HSV-1 lacking the immunodominant gB498-505 epitope (S1L). S1L induced a numerically equivalent CD8+ T cell infiltrate in the TG that was HSV-specific, but lacked specificity for gB498-505. Instead, there was a general increase of non-gB-CD8s with specific subdominant epitopes arising to codominance. In a latent S1L infection, non-gB-CD8s in the TG showed a hierarchy targeting different epitopes at latency compared to at acute times, and these cells retained an increased functionality at latency. In a latent S1L infection, these non-gB-CD8s also display an equivalent ability to block HSV reactivation in ex vivo ganglionic cultures compared to TG infected with wild type HSV-1. These data indicate that loss of the immunodominant gB498-505 epitope alters the dominance hierarchy and reduces functional compromise of CD8+ T cells specific for subdominant HSV-1 epitopes during viral latency.

The evaluation of diagnostic efficiency for stromal herpes simplex keratitis by the combination of tear HSV-sIgA and HSV-DNA.

PURPOSE: To assess the differential diagnostic values for stromal herpes simplex keratitis (HSK) by using tear HSV-sIgA, tear HSV-DNA, and the combination. METHODS: Tear samples for both eyes and the paired serum were collected from 187 stromal HSK and 56 controls. Enzyme-linked immune sorbent assay (ELISA) was used to analyze the tear HSV-sIgA and serum IgG/IgM/IgA. The levels of tear HSV-DNA were measured by polymerase chain reaction (PCR). RESULTS: The positive rates for tear HSV-sIgA and HSV-DNA were 36.90% and 10.96% respectively in stromal HSK patients. Twelve showed positivity for both sIgA and DNA, while 46 cases were positive for sIgA or DNA. The sensitivity, specificity, PPV, and NPV for simultaneous measurement were 39.73%, 98.21%, 98.31%, and 38.46%. The total negative conversion rate of sIgA was 95.71%. CONCLUSIONS: The diagnostic efficiency of HSV-sIgA only is nearly equal to the combination of HSV-sIgA and HSV-DNA, and the positive result is optimum to achieve a reliable diagnosis of stromal HSK even in atypical or unsuspected cases.

Α molecular epidemiological analysis of adenoviruses from excess conjunctivitis cases.

BACKGROUND: Τo perform a molecular epidemiological analysis of viral conjunctivitis among excess conjunctivitis cases recorded at the University Hospital of Patras, Greece, for the period March to June 2012. METHODS: A structured questionnaire containing demographic and clinical data was developed in order to collect retrospective data on the cases. Eye swab specimens were collected and molecular detection of adenoviruses was performed by nested PCR. Positive results were confirmed by sequencing. To determine the relatedness between the isolated sequences, a phylogenetic analysis was conducted. RESULTS: The epidemiological analysis (including retrospective data) included 231 conjunctivitis cases (47.1% male, and 52.8% female). Based on clinical features 205 of the cases were diagnosed of viral origin (46.3% male and 53.7% female), 4 of bacterial origin (50% male and 50% female) while 22 were undefined conjunctivitis. The outbreak excess cases (included 156 cases) affected all age groups regardless gender predilection. For the positive samples indicated that 29 samples (72.5%) were AdV17, and 5 (12.5%) as AdV54. CONCLUSIONS: Molecular analysis could define the cause of viral conjunctivitis, while epidemiological data contributed to the assessment of the risk factors and underlined possible preventive measures. This study is one of the very few on viral conjunctivitis in Greece. This outbreak underscores the need for a national surveillance system for acute infectious conjunctivitis outbreaks. The epidemiological as well as molecular investigation on HAdV ocular infections is rather absent in Greece, which has no surveillance system for viral conjunctivitis.

Bilateral tear film alterations in patients with unilateral quiescent herpes simplex keratitis.

PURPOSE: To evaluate bilateral tear function and corneal sensitivity in patients with unilateral quiescent herpes simplex keratitis (HSK) and determine the correlation between corneal sensitivity and tear secretion in both eyes. METHODS: Thirty-five patients diagnosed with unilateral quiescent HSK and 35 heathy controls were included in this study. Bilateral tear osmolarity, Schirmer's test, the tear break-up time (TBUT) and corneal sensitivity were measured in all participants. RESULTS: In the HSK group, both eyes demonstrated a significant increase in tear osmolarity, and a decrease in Schirmer's test and the TBUT compared with healthy controls (All p < 0.001). The bilateral tear osmolarity and Schirmer's test were similar, but the TBUT (4.9 ± 2.1 versus 7.4 ± 2.0 second; p < 0.001) and corneal sensitivity (35.1 ± 1.9 versus 54.3 ± 0.8 mm; p < 0.001) were significantly lower in the affected eyes. The bilateral tear osmolarity, Schirmer's test and the TBUT were significantly correlated with corneal sensitivity of the affected eye (All p < 0.001). When corneal sensitivity of the unaffected eye was treated as a control variable, tear osmolarity (R = -0.626, p < 0.001), Schirmer's test (R = 0.739, p < 0.001) and the TBUT (R = 0.691, p < 0.001) of the unaffected eyes were still significantly correlated with the corneal sensitivity of the affected eyes. CONCLUSIONS: Unilateral quiescent HSK causes bilateral tear impairment, which depends on the loss of corneal sensitivity in the affected eye. In the affected eye with severe corneal sensitivity loss, bilateral dry eye occurred.