VIRUS INFECTIONS IN THE EYE

我们对各种病毒学和 当病毒和寄生虫发生的免疫病理过程 眼部微环境中的复制包括五个方面：(1) 病毒诱导的视网膜变性过程；(2)可能的作用 病毒在人类疾病中的研究；(3)分子诊断和 人巨细胞病毒感染的发病机制；(4) 水痘带状疱疹病毒(VZV)视网膜感染和(5) 弓形虫对视网膜的感染。我们已经确立了 小鼠冠状病毒可以诱发眼部疾病，并可用于 作为研究视网膜退行性疾病的模型系统。这 模型有许多独特的功能。该病毒能够诱导一种 急性感染时伴有轻度视网膜血管炎症。 最初的视网膜损伤之后是传染性病毒的清除 和进行性视网膜变性。这是第一个视网膜模型 为了证明病毒引起的退化，病毒的持久性， 对病毒引起的组织损伤和病毒的遗传易感性 触发了自身免疫反应。在过去的一年里，我们有 评估了细胞凋亡在这种视网膜疾病中的作用。数字 在感染的眼睛中，凋亡细胞的数量显著增加， 在第6天到第10天达到最大值。双标 免疫组织化学染色显示，细胞内的凋亡事件 视网膜与病毒抗原和CD8+T细胞相关。RT- 聚合酶链式反应分析证实Fas配体(FasL)和 感染视网膜内颗粒酶B的mRNAs。最新进展 细胞凋亡、调节基因表达和病毒清除 两者的视网膜变性易感性(BALB/c)和 抗性(CD-1)小鼠。因此，细胞凋亡与视网膜有关。 炎症和传染性病毒的减少。体外研究 证明JHM病毒不能直接诱导细胞凋亡 被感染的细胞。这些发现表明，视网膜细胞凋亡是 由病毒-宿主相互作用触发，然后可能导致 限制视网膜感染。人类巨细胞病毒是一种疱疹病毒 先天性失明儿童的主要致盲原因 感染和免疫功能受损的个体。很难做到 研究人类巨细胞病毒潜伏期。因此建立了CMV的细胞培养模型。 复制和延迟可能会让您深入了解 另类治疗方式。为了了解视网膜 对于CMV的组织嗜性，我们扩展了我们最初的研究 HRPE中的CMV复制。数据表明，对 人视网膜色素上皮细胞的巨细胞病毒感染与人不同 在病毒进入和转录水平上的成纤维细胞 病毒基因组。此外，数据表明，HCMV使用 硫酸肝素结合和Gh-GL糖蛋白复合体作为 单元格输入的方式。CMV宿主细胞的这些差异 HRPE细胞中的相互作用可能在CMV中起作用 在眼睛内激活、复制和扩散。在一个单独的系列中 我们已经对诊断和治疗方法进行了评估 巨细胞病毒感染。这些研究证明了这种巨细胞病毒的实用性。 RPE细胞模型系统用于评估病毒复制和疗效 使用反义寡核苷酸进行抗病毒治疗。巨细胞病毒感染 相关疾病仍然是一个主要的并发症。 在骨髓移植患者中遇到。我们发现 流式细胞仪鉴定巨细胞病毒抗原血症与 移植患者中的病毒分离，并可能成为预测 血液中CMV的快速检测。人VZV感染的 眼睛可能导致角膜炎、葡萄膜炎、急性视网膜坏死或 艾滋病患者进行性视网膜外坏死。然而，a 复制这些疾病的小动物模型还不存在。 我们已经证明，玻璃体内接种豚鼠 VZV导致由单个核细胞组成的慢性葡萄膜炎 渗入眼后段。使用重组体 表达β-半乳糖苷酶的VZV，我们检测到 这种酶在眼内植入后长达四个月 接种疫苗。在过去的一年里，我们利用这一模式 评估VZV疫苗。我们发现，VZV GE免疫 和GI重组蛋白诱导强大的体液和细胞 加速清除VZV DNA的反应，可能 中和眼睛内的病毒。

Our studies of various virologic and immunopathologic processes that occur when viruses and parasites replicate in the ocular microenvironment comprise five 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; (4) Varicella - zoster virus (VZV) infections of the retina and (5) Toxoplasma gondii infections of the retina. We have established that murine coronavirus can induce ocular disease and may be used as a model system for studying retinal degenerative diseases. This model has many unique features. 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. During the past year we have evaluated the role of apoptosis in this retinal disease. The number of apoptotic cells was significantly elevated in infected eyes, reaching a maximum at days 6 to 10. Double-labeled immunostaining demonstrated that apoptotic events within the retina were associated with viral antigen and CD8+ T cells. RT- PCR analysis identified the upregulation of Fas ligand (FasL) and granzyme B mRNAs within the infected retinas. The development of apoptosis, regulative gene expression and viral clearance were similar, in both, retinal degeneration susceptible (BALB/c) and resistant (CD-1) mice. Thus, apoptosis was associated with retinal inflammation and a decrease of infectious virus. In vitro studies demonstrated that JHM virus did not directly induce apoptosis in infected cells. These findings indicated that retinal apoptosis was triggered by virus-host interactions which then may contribute to limit the retinal infection. Human CMV is a herpesvirus that is a major cause of blindness in children born with congenital infections and in immunocompromised individuals. It is difficult to study CMV latency in man. Therefore cell culture models of CMV replication and latency may provide insight into a rationale for alternative treatment modalities. In order to understand the retinal tissue tropism for CMV, we have extended our original studies of CMV replication in HRPE . The data indicate that regulation of HCMV infection of HRPE cells differed from that of human fibroblasts at both the levels of virus entry and transcription of the viral genome. Moreover, the data demonstrate that HCMV uses heparin sulfate binding and the gH ?gL glycoprotein complex as a means of cell entry. These differences in CMV - host cell interactions in HRPE cells which may be instrumental in CMV activation, replication and spread within the eye. In a separate series of studies we have evaluated methods of diagnosis and treatment of CMV infections. These studies demonstrate the utility of this CMV RPE cell model system to evaluate virus replication and efficacy of antiviral therapy with antisense oligonucleotides. CMV infection and associated diseases continue to be a major complication encountered in bone marrow transplant patients. We found that flow cytometric identification of CMV antigenemia correlates with virus isolation in transplant patients and may be a predictive test for the rapid detection of CMV in the blood. Human VZV infections of the eye can result in keratitis, uveitis, acute retinal necrosis or progressive outer retinal necrosis in AIDS patients. However, a small animal model that replicates these diseases does not yet exist. We have shown that intravitreal inoculation of guinea pigs with VZV results in a chronic uveitis consisting of a mononuclear cell infiltrate in the posterior segment of the eye. Using a recombinant VZV that expresses beta-galactosidase, we detected expression of the enzyme in the eye for up to four months after intraocular inoculation. During the past year we have utilized this model to evaluate VZV vaccines. We found that immunization with VZV gE and gI recombinant proteins induced potent humoral and cellular responses that accelerated the clearance of VZV DNA and may neutralize virus within the eye.