PURPOSE. The rd12 mouse was reported as a recessively inherited Rpe65 mutation. We asked if the rd12 mutation resides in Rpe65 and how the mutation manifests itself.
METHODS. A complementation test was performed by mating Rpe65KO (KO/KO) and rd12 mice together to determine if the rd12 mutation is in the Rpe65 gene. Visual function of wildtype (+/+), KO/+, rd12/+, KO/KO, rd12/rd12, and KO/rd12 mice was measured by optokinetic tracking (OKT) and ERG. Morphology was assessed by retinal cross section. qRTPCR quantified Rpe65 mRNA levels. Immunoblotting measured the size and level of RPE65 protein. Rpe65 mRNA localization was visualized with RNA fluorescence in situ hybridization (FISH). Fractions of Rpe65 mRNA-bound proteins were separated by linear sucrose gradient fractionation.
RESULTS. The KO and rd12 alleles did not complement. The rd12 allele induced a negative semidominant effect on visual function; OKT responses became undetectable 120 days earlier in rd12/rd12 mice compared with KO/KO mice. rd12/+ mice lost approximately 21% visual acuity by P210. rd12/rd12 mice had fewer cone photoreceptor nuclei than KO/KO mice at P60. rd12/rd12 mice expressed 71% +/+ levels of Rpe65 mRNA, but protein was undetectable. Mutant mRNA was appropriately spliced, exported to the cytoplasm, trafficked, and contained no other coding mutation aside from the known nonsense mutation. Mutant mRNA was enriched on ribosome-free messenger ribonucleoproteins (mRNPs), whereas wildtype mRNA was enriched on actively translating polyribosomes.
CONCLUSIONS. The rd12 lesion is in Rpe65. The rd12 mutant phenotype inherits in a semidominant manner. The effects of the mutant mRNA on visual function may result from inefficient binding to ribosomes for translation.
by
Jessica Shantha;
John G. Mattia;
Augustine Goba;
Kayla G. Barnes;
Faiqa K. Ebrahim;
Colleen S Kraft;
Brent R. Hayek;
Jessica N. Hartnett;
Jeffrey G. Shaffer;
John S. Schieffelin;
John D. Sandi;
Mambu Momoh;
Simbirie Jalloh;
Donald S. Grant;
Kerry Dierberg;
Joyce Chang;
Sharmistha Mishra;
Adrienne K. Chan;
Rob Fowler;
Tim O'Dempsey;
Erick Kaluma;
Taylor Hendricks;
Roger Reiners;
Melanie Reiners;
Lowell A. Gess;
Kwame ONeill;
Sarian Kamara;
Alie Wurie;
Mohamed Mansaray;
Nisha R. Acharya;
William J. Liu;
Sina Bavari;
Gustavo Palacios;
Moges Teshome;
Ian Crozier;
Paul E. Farmer;
Timothy M. Uyeki;
Daniel G. Bausch;
Robert F. Garry;
Matthew J. Vandy;
Steven Yeh
Background: Ebola virus disease (EVD) survivors are at risk for uveitis during convalescence. Vision loss has been observed following uveitis due to cataracts. Since Ebola virus (EBOV) may persist in the ocular fluid of EVD survivors for an unknown duration, there are questions about the safety and feasibility of vision restorative cataract surgery in EVD survivors. Methods: We conducted a cross-sectional study of EVD survivors anticipating cataract surgery and patients with active uveitis to evaluate EBOV RNA persistence in ocular fluid, as well as vision outcomes post cataract surgery. Patients with aqueous humor that tested negative for EBOV RNA were eligible to proceed with manual small incision cataract surgery (MSICS). Findings: We screened 137 EVD survivors from June 2016 – August 2017 for enrolment. We enrolled 50 EVD survivors; 46 with visually significant cataract, 1 with a subluxated lens, 2 with active uveitis and 1 with a blind painful eye due to uveitis. The median age was 24.0 years (IQR 17–35) and 35 patients (70%) were female. The median logMAR visual acuity (VA) was 3.0 (Snellen VA Hand motions; Interquartile Range, IQR: 1.2-3.0, Snellen VA 20/320 – Hand motions). All patients tested negative for EBOV RNA by RT-PCR in aqueous humor/vitreous fluid and conjunctiva at a median of 19 months (IQR 18-20) from EVD diagnosis in Phase 1 of ocular fluid sampling and 34 months (IQR 32-36) from EVD diagnosis in Phase 2 of ocular fluid sampling. Thirty-four patients underwent MSICS, with a preoperative median VA improvement from hand motions to 20/30 at three-month postoperative follow-up (P < 0.001). Interpretation: EBOV persistence by RT-PCR was not identified in ocular fluid or conjunctivae of fifty EVD survivors with ocular disease. Cataract surgery can be performed safely with vision restorative outcomes in patients who test negative for EBOV RNA in ocular fluid specimens. These findings impact the thousands of West African EVD survivors at-risk for ocular complications who may also require eye surgery during EVD convalescence.
Herpes simplex virus type 1 (HSV1) remains one of the most ubiquitous human pathogens on earth. The classical presentation of HSV1 infection occurs as a recurrent lesions of the oral mucosa commonly refer to as the common cold sore. However, HSV1 also is responsible for a range of ocular diseases in immunocompetent persons that are of medical importance, causing vision loss that may result in blindness. These include a recurrent corneal disease, herpes stromal keratitis, and a retinal disease, acute retinal necrosis, for which clinically relevant animal models exist. Diverse host immune mechanisms mediate control over herpesviruses, sustaining lifelong latency in neurons. Programmed cell death (PCD) pathways including apoptosis, necroptosis, and pyroptosis serve as an innate immune mechanism that eliminates virus-infected cells and regulates infection-associated inflammation during virus invasion. These different types of cell death operate under distinct regulatory mechanisms but all server to curtail virus infection. Herpesviruses, including HSV1, have evolved numerous cell death evasion strategies that restrict the hosts ability to control PCD to subvert clearance of infection and modulate inflammation. In this review, we discuss the key studies that have contributed to our current knowledge of cell death pathways manipulated by HSV1 and relate the contributions of cell death to infection and potential ocular disease outcomes.
by
Justine R. Smith;
Shawn Todd;
Liam M. Ashander;
Theodosia Charitou;
Yuefang Ma;
Steven Yeh;
Ian Crozier;
Michael Z. Michael;
Binoy Appukuttan;
Keryn A. Williams;
David J. Lynn;
Glenn A. Marsh
PURPOSE: Success of Ebola virus (EBOV) as a human pathogen relates at the molecular level primarily to blockade the host cell type I interferon (IFN) antiviral response. Most individuals who survive Ebola virus disease (EVD) develop a chronic disease syndrome: approximately one-quarter of survivors suffer from uveitis, which has been associated with presence of EBOV within the eye. Clinical observations of post-Ebola uveitis indicate involvement of retinal pigment epithelial cells.
METHODS: We inoculated ARPE-19 human retinal pigment epithelial cells with EBOV, and followed course of infection by immunocytochemistry and measurement of titer in culture supernatant. To interrogate transcriptional responses of infected cells, we combined RNA sequencing with in silico pathway, gene ontology, transcription factor binding site, and network analyses. We measured infection-induced changes of selected transcripts by reverse transcription-quantitative polymerase chain reaction. RESULTS: Human retinal pigment epithelial cells were permissive to infection with EBOV, and supported viral replication and release of virus in high titer. Unexpectedly, 28% of 560 upregulated transcripts in EBOV-infected cells were type I IFN responsive, indicating a robust type I IFN response. Following EBOV infection, cells continued to express multiple immunomodulatory molecules linked to ocular immune privilege.
CONCLUSIONS: Human retinal pigment epithelial cells may serve as an intraocular reservoir for EBOV, and the molecular response of infected cells may contribute to the persistence of live EBOV within the human eye.
TRANSLATIONAL RELEVANCE: This bedside-to-bench research links ophthalmic findings in survivors of EVD who suffer from uveitis with interactions between retinal pigment epithelial cells and EBOV.
by
Shreyasi Choudhury;
Christianne E. Strang;
John Alexander;
Miranda L. Scalabrino;
Julie Lynch Hill;
Daniel T. Kasuga;
C. Douglas Witherspoon;
Sanford L. Boye;
Paul D. Gamlin;
Shannon E. Boye
Purpose: The ability to generate macaque retinas with sortable cell populations would be of great benefit to both basic and translational studies of the primate retina. The purpose of our study was therefore to develop methods to achieve this goal by selectively labeling, in life, photoreceptors (PRs) and retinal ganglion cells (RGCs) with separate fluorescent markers.
Methods: Labeling of macaque (Macaca fascicularis) PRs and RGCs was accomplished by subretinal delivery of AAV5-hGRK1-GFP, and retrograde transport of micro-ruby™ from the lateral geniculate nucleus, respectively. Retinas were anatomically separated into different regions. Dissociation conditions were optimized, and cells from each region underwent fluorescent activated cell sorting (FACS). Expression of retinal cell type- specific genes was assessed by quantitative real-time PCR to characterize isolated cell populations.
Results: We show that macaque PRs and RGCs can be simultaneously labeled in-life and enriched populations isolated by FACS. Recovery from different retinal regions indicated efficient isolation/enrichment for PRs and RGCs, with the macula being particularly amendable to this technique.
Conclusions: The methods and materials presented here allow for the identification of novel reagents designed to target RGCs and/or photoreceptors in a species that is phylogenetically and anatomically similar to human. These techniques will enable screening of intravitreally-delivered AAV capsid libraries for variants with increased tropism for PRs and/or RGCs and the evaluation of vector tropism and/or cellular promoter activity of gene therapy vectors in a clinically relevant species.
Recent Ebola epidemics, the ongoing COVID-19 pandemic, and emerging infectious disease threats have highlighted the importance of global infectious diseases and responses to public health emergencies. Ophthalmologists are essential health care workers who provide urgent and emergent vision care services during outbreaks and address the ocular consequences of epidemic and pandemic infectious diseases. In 2017, theWorld Health Organization (WHO) identified high priority pathogens likely to cause a future epidemic with the goal of guiding research and development to improve diagnostic tests, vaccines, and medicines. These measures were necessary to better inform and respond to public health emergencies.
Given the ocular complications associated with emerging infectious diseases, there is a need to recognize the ophthalmic sequelae for future vision health preparedness for potential future outbreaks. The WHO High Priority pathogens list provides a roadmap for ophthalmologists and subspecialty providers that will guide strategic areas of research for clinical care and preparedness for future pandemic threats. This review summarizes these key viral pathogens, summarizes major systemic disease findings, and delineates relevant ocular complications of the WHO High Priority pathogens list, including Crimean-Congo hemorrhagic fever, Filovirus diseases (Ebola virus disease and Marburg hemorrhagic fever), human Coronaviruses, Lassa Fever, Nipah virus infection, Zika, and Rift Valley fever.
Introduction:
The largest Ebola virus (EBOV) outbreak occurred from 2013 – 2016 in West Africa and consequently resulted in the largest cohort of Ebola virus disease (EVD) survivors to date. Ocular disease is among the most common sequelae reported in EVD survivors. This review discusses the prevalence, manifestations, pathogenesis, diagnosis and management of EVD-related ocular disease.
Areas covered:
An extensive review of the literature was performed to detail the prevalence and manifestations of EVD-related ocular disease. We also review current eye screening and treatment strategies and our current understanding and approach to invasive ophthalmic procedures including surgery.
Expert opinion:
The ocular sequelae of EVD can lead to vision impairment or blindness, if untreated. Keys to the prevention of such an outcome include timely evaluation and access to appropriate ophthalmic care. The persistence of EBOV in the eye and other immune-privileged sites is the subject of ongoing investigation, but should not be a barrier to care if appropriate screening and biosafety measures are taken. Improved understanding of the pathogenesis of this condition and ongoing clinical care are needed for EVD survivors at-risk for ocular complications.
by
Christopher D. Bowen;
Daniel W. Renner;
Jacob T Shreve;
Yolanda Tafuri;
Kimberly M. Payne;
Richard Dix;
Paul R. Kinchington;
Derek Gatherer;
Moriah L. Szpara
Herpes simplex virus 1 (HSV-1) is a widespread global pathogen, of which the strain KOS is one of the most extensively studied. Previous sequence studies revealed that KOS does not cluster with other strains of North American geographic origin, but instead clustered with Asian strains. We sequenced a historical isolate of the original KOS strain, called KOS63, along with a separately isolated strain attributed to the same source individual, termed KOS79. Genomic analyses revealed that KOS63 closely resembled other recently sequenced isolates of KOS and was of Asian origin, but that KOS79 was a genetically unrelated strain that clustered in genetic distance analyses with HSV-1 strains of North American/European origin. These data suggest that the human source of KOS63 and KOS79 could have been infected with two genetically unrelated strains of disparate geographic origins. A PCR RFLP test was developed for rapid identification of these strains.
by
Duncan E Berry;
Clay J Bavinger;
Alcides Filho Fernandes;
John Mattia;
Jalika Mustapha;
Lloyd Harrison-Williams;
Moges Teshome;
Matthew J Vandy;
Jessica Shantha;
Steven Yeh
The largest Ebola virus disease (EVD) outbreak occurred from 2013–2016 in West Africa, leading to over 28,600 cases and 11,300 deaths and resulted in the largest cohort of EVD survivors to date.1 Another recent outbreak in Democratic Republic of the Congo (DRC) has resulted in 3,481 cases with over 1,170 survivors since August 2018.2 In studies of large EVD survivor cohorts, uveitis is the most common ocular manifestation with a 13–34% reported prevalence.3,4 Cataract, with or without uveitis, is the second most common finding and has been reported in 10% of EVD survivors.3,4 The Ebola Virus Persistence in Ocular Tissues (EVICT) study was a cross-sectional study which supported the safety of cataract surgery in EVD survivors5, but may not be generalizable to vitreoretinal surgery. Posterior segment findings in EVD survivors have been described primarily in relationship to uveitis and include vitritis, chorioretinitis and chorioretinal scarring.3,6 The prevalence of posterior segment findings in EVD survivors, particularly those that may require surgical intervention, is of particular interest given the risk of Ebola virus (EBOV) persistence in ocular tissues and fluids.7
by
Jessica Shantha;
Dominick Canady;
Caleb Hartley;
Amy Cassedy;
Chris Miller;
Sheila T Angeles-Han;
Lloyd CM Harrison-Williams;
Matthew J Vandy;
Natalie Weil;
Gilberte Bastien;
Steven Yeh
Background: Ebola virus disease (EVD) outbreaks in West Africa (2013-2016) and the Democratic Republic of Congo (2018-2020) have resulted in thousands of EVD survivors who remain at-risk for survivor sequelae. While EVD survivorship has been broadly reported in adult populations, pediatric EVD survivors are under-represented. In this cross-sectional study, we investigated the prevalence of eye disease, health-related quality-of-life, vision-related quality-of-life, and the burden of mental illness among pediatric EVD survivors in Sierra Leone. Methods: Twenty-three pediatric EVD survivors and 58 EVD close contacts were enrolled. Participants underwent a comprehensive ophthalmic examination and completed the following surveys: Pediatric Quality of Life Inventory Version 4.0, Effect of Youngsters Eyesight on Quality-of-Life, and the Revised Child Anxiety and Depression Scale. Findings: A higher prevalence of uveitis was observed in EVD survivor eyes (10·8%) cohort compared to close contacts eyes (1·7%, p=0·03). Overall, 47·8% of EVD survivor eyes and 31·9% of close contact eyes presented with an eye disease at the time of our study (p=0·25). Individuals diagnosed with an ocular complication had poorer vision-related quality-of-life (p=0·02). Interpretation: Both health related quality-of-life and vision-related quality-of-life were poor among EVD survivors and close contacts. The high prevalence of eye disease associated with reduced vision health, suggests that cross-disciplinary approaches are needed to address the unmet needs of EVD survivors. Funding: National Institutes of Health R01 EY029594, K23 EY030158; National Eye Institute; Research to Prevent Blindness (Emory Eye Center); Marcus Foundation Combating Childhood Illness; Emory Global Health Institute; Stanley M. Truhlsen Family Foundation.