Pathological mechanisms of serious and neglected zoonotic retinal infections

Author: Genevieve Oliver

Oliver, Genevieve, 2021 Pathological mechanisms of serious and neglected zoonotic retinal infections, Flinders University, College of Medicine and Public Health

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Zoonotic diseases form the bulk of emerging infections, and are responsible for epidemics and pandemics that impact global health and security. Many of these pathogens cause ocular disease, and retinal involvement often results in visual impairment. The retina is a complex tissue that has limited capacity to regenerate. It performs a critical sensory function, converting the visual image into a neural signal in the initial stages of visual processing. This thesis studied three important zoonoses that involve the retina: Toxoplasma gondii, dengue virus (DENV), and Ebola virus. The ubiquitous parasite T. gondii has infected one third of the human population, and retinal involvement results in tissue destruction with resultant visual impairment. The cat is the definitive host, and mammals and birds are intermediate hosts. Each of the four serotypes of DENV, a mosquito-borne epidemic flavivirus, causes a serious tropical disease. The features of retinal involvement in dengue infection are diverse and are poorly understood. The largest Ebola epidemics have been caused by Ebola virus spill-over from the bat population. Survivors develop a multisystem syndrome that includes infectious ocular pathology and serious visual sequelae.

This thesis advances the body of knowledge of retinal infections on many fronts. Spectral-domain optical coherence tomography (SD-OCT) is considered a standard tool in clinical ophthalmology that renders in vivo, real-time data on tissue architecture. Through the largest study of toxoplasmic retinochoroiditis by SD-OCT, this work presents a thorough portrayal of the spectrum of the tissue-level changes that occur as a result of infection. The investigation identified signs that were characteristic of the disease and/or indicated poor visual prognosis. The presence of retinal hyporeflective spaces or signal voids, likely representing liquefactive necrosis of the retina, was identified as an SD-OCT sign associated with visual acuity of worse than 20/200 (defined as legal blindness in Australia and other industrialised nations, including the United States). This observation is expected to be useful to the practising ophthalmologist.

This thesis also presents the first published work on human Müller cells in DENV infection, and provides first details of the molecular responses to DENV. Müller cells are a specialized glial cell unique to the retina. They form part of the blood-retinal barrier, contact most other retinal cell populations, and provide critical physiological support to neurons. To understand the response of human Müller cells to infection in dengue, the MIO-M1 human Müller cell line and primary human Müller cell isolates were infected with multiple DENV strains of DENV1 and DENV2 serotypes, and antiviral, inflammatory and immunomodulatory responses after DENV infection were evaluated. Müller cells mounted an inflammatory response to DENV infection that was characterised by upregulation of pro-inflammatory cytokines, and the response varied by viral strain, with DENV2 inducing stronger responses. Curiously, a lack of a type I interferon response to infection was demonstrated in MIO-M1 cells, but not primary human Müller isolates, which this thesis is the first to report.

The pathogenesis of post-Ebola uveitis is poorly understood. Viral persistence in the eye, despite clinical recovery, has been demonstrated, and pigmented retinal scars in survivors implicate involvement of retinal pigment epithelial cells. To understand the potential involvement of microRNA (miRNA) signalling in EBOV infection of human retinal pigment epithelial cells, bioinformatic processes were utilized to predict pathways and networks based on miRNA expression in the ARPE-19 human retinal pigment epithelial cell line following infection with EBOV. Bioinformatic analysis of miRNA-driven pathways in EBOV-infected ARPE-19 cells identified leucine-rich repeat kinase (LRRK) 2, a protein associated with neuroinflammation, as the most highly connected molecule in miRNA signalling networks that were active in EBOV-infected retinal pigment epithelial cells. Mitogen-activated protein kinases (MAPK) 13 and 7 were also highly involved in EBOV infection and are associated with neuroinflammation. Central to the response to EBOV infection, miR-190a and miR-101 may facilitate EBOV survival in retinal pigment epithelium through prevention of apoptosis.

To examine the effect of the EBOV protein, VP35, on the type I interferon (IFN) antiviral response by human retinal pigment epithelial cells, a plasmid expressing VP35 was transfected into the ARPE-19 cell line and primary cell isolates. Expression of VP35 inhibited IFN-β production in some primary human retinal pigment epithelial cell isolates, indicating that this viral protein plays a key role in subversion of the host cell, and this interaction varies across individuals. These mechanistic studies of the interplay between EBOV and the type I IFN response in retinal pigment epithelial cells represent first-in-field experiments.

The overall aim of this thesis was to identify and address unanswered questions relating to the mechanisms of retinal pathology caused by three serious human pathogens that are transmitted by animals: T. gondii, DENV and EBOV. Various aspects of the host cellular and/or molecular response to an infection with each of these three pathogens were addressed in this thesis. The work has demonstrated a range of indicators of retinal tissue damage and provides a foundation for further mechanistic research and potential therapeutic opportunities to reduce infection-mediated tissue damage and resultant visual impairment in zoonotic retinal infections.

Keywords: Zoonoses, retina, infection, Toxoplasma gondii, parasite, dengue virus, Ebola virus, virology, Müller cells, retinal pigment epithelium, biomarkers, optical coherence tomography, molecular biology, bioinformatics.

Subject: Medicine thesis

Thesis type: Doctor of Philosophy
Completed: 2021
School: College of Medicine and Public Health
Supervisor: Justine Smith