Virus Infections In The Eye

我们对病毒和寄生虫在眼微环境中复制时发生的各种病毒学和免疫病理过程的研究包括四个方面：(1)病毒诱导的视网膜退行性过程；(2)病毒在人类疾病中的可能作用；(3)人巨细胞病毒（CMV）感染的分子诊断与发病机制；(4)眼部疱疹病毒感染。我们建立了研究视网膜退行性疾病的模型系统——实验性冠状病毒视网膜病变（ECOR）。该病毒能够在轻度视网膜血管炎症的情况下诱发急性感染。最初的视网膜损伤随后是感染性病毒的清除和进行性视网膜变性。这是第一个证明病毒诱导变性、病毒持久性、病毒诱导组织损伤的遗传易感性和病毒引发自身免疫反应的视网膜模型。我们的目标是确定病理生理机制和识别基因参与视网膜退行性疾病。在过去一年中，我们取得了以下主要成果。基于动物模型系统，我们也开始了评估人类视网膜退行性疾病的研究。采用免疫细胞化学染色和免疫印迹法检测不明原因视网膜病变患者的自身抗体。我们鉴定了三例锥体棒变性患者，其抗体高滴度针对神经节细胞和内核层。在已知遗传性视网膜变性、葡萄膜炎患者或正常人的血清中未检测到这些水平的抗视网膜反应性。从锥杆变性患者血清视网膜cDNA文库中鉴定出LEDGF和ATR-X两种抗原。3例锥杆变性患者血清均表现出LEDGF的反应性。特异性抗视网膜抗体的鉴定有助于改变视网膜细胞功能和活力，可能为视网膜细胞功能障碍的机制提供新的见解。

Our studies of various virologic and immunopathologic processes that occur when viruses and parasites replicate in the ocular microenvironment comprise four areas: (1) virus induced retinal degenerative processes; (2) the possible roles of viruses in human diseases; (3) molecular diagnosis and pathogenesis of cytomegalovirus (CMV) infections in man; and (4) herpesvirus infections of the eye. We have established a model system for studying retinal degenerative diseases, experimental coronavirus retinopathy (ECOR). The virus is capable of inducing an acute infection in the presence of mild retinal vascular inflammation. Initial retinal damage is followed by clearance of infectious virus and progressive retinal degeneration. This is the first retinal model to demonstrate a virus induced degeneration, viral persistence, a genetic predisposition to virus induced tissue damage and a virus triggered autoimmune response. Our goal is to determine the pathophysiological mechanisms and to identify genes involved in the retinal degenerative disease. During the past year we have made the following key findings. Based on the animal model system, we have also initiated studies to evaluate human retinal degenerative diseases. Autoantibodies were detected in patients with retinopathy of unknown origin by immunocytochemical staining and western blot analysis. We have identified three patients with cone-rod degeneration with high titers of antibody directed against the ganglion cells and inner nuclear layer. Anti-retinal reactivity at these levels were not detected in sera from patients with known genetic retinal degenerations, uveitis or from normal individuals. Two antigens, LEDGF and ATR-X, were identified from a retina cDNA library with sera from a cone-rod degeneration patient. The sera of all three cone-rod degeneration patients demonstrated reactivity to LEDGF. Identification of the specific anti-retinal antibodies that contribute to altered retinal cell function and viability may provide new insights into mechanisms of retinal cell dysfunction. Corneal herpes infection elicits a robust inflammatory response and eventually leads to a vision-threatening stromal keratitis as a sequela of frequent reactivation of latent virus. The aetiology of herpetic stromal keratitis is thought to be an aberrant Th1 cytokine mediated immunopathology. We evaluated HSV infection in human corneal epithelial and corneal fibroblast cells. We found that HSV DNA resulted in augmented TLR-3 and 9 gene expression and IL-6 release. As virus is frequently in the form of neutralized virus immune complexes, the ability of these immune complexes to interact with TLRs and trigger IL-6 production was evaluated. We found that HSV-anti-HSV IgG complexes were as potent as HSV DNA in their ability to induce IL-6. These phenomena were mediated via augmented TLR-3 and .9 gene expression. These studies indicate that viral DNA and/or immune complexes deposited in corneal stroma may continue to trigger stromal inflammation by excessive cytokine release via TLR3 and 9 long after the initial acute viral insult has subsided. Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. We evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and determined their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-kB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (P< 0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and Real Time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses.