VIRUS INFECTIONS IN THE EYE

我们对病毒和寄生虫在眼睛微环境中复制时发生的各种病毒学和免疫病理学过程的研究包括五个方面：(1)病毒诱导的视网膜退化过程；(2)病毒在人类疾病中的可能作用；(3)巨细胞病毒(CMV)感染的分子诊断和发病机制；(4)水痘-带状疱疹病毒(VZV)感染眼部和(5)弓形虫感染视网膜。我们建立了一个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(ECR)。该病毒能够在存在轻度视网膜血管炎症的情况下引发急性感染。最初的视网膜损伤之后是感染性病毒的清除和进行性的视网膜变性。这是第一个显示病毒引起的变性、病毒持久性、对病毒引起的组织损伤的遗传易感性和病毒引发的自身免疫反应的视网膜模型。我们的目标是确定视网膜退行性疾病的病理生理机制，并确定与之相关的基因。在过去的一年里，我们评估了细胞因子的作用。眼内细胞因子水平分析显示，ECR患者IL-1、IL-6、肿瘤坏死因子-α和干扰素-γ水平升高。干扰素-g和颗粒介导的细胞毒性淋巴细胞(CL)在限制病毒传播方面发挥着重要作用，并可能在免疫病理中起作用。免疫细胞化学染色检测CD8T细胞，RT-PCR法检测细胞毒颗粒、颗粒酶B。视网膜内干扰素-g和颗粒酶B基因表达的序列分析表明，在清除视网膜感染病毒时，这些基因表达上调。这些研究表明，细胞毒性T细胞和T细胞产物(干扰素-g和颗粒酶B)存在于视网膜内，有助于清除视网膜病毒。人类巨细胞病毒是一种疱疹病毒，是先天性感染儿童和免疫功能受损儿童失明的主要原因。人类巨细胞病毒潜伏期的研究比较困难。因此，CMV复制和潜伏期的细胞培养模型可能为替代治疗方式提供理论基础。为了了解CMV在视网膜组织中的趋向性，我们对CMV在hRPE中复制的原始研究进行了扩展。结果表明，人RPE细胞对HCMV感染的调控在病毒进入和病毒基因组转录水平上与人成纤维细胞不同。在hRPE细胞中，CMV与宿主细胞相互作用的这些差异可能有助于CMV的激活、复制和在眼睛内传播。在另一系列研究中，我们评估了巨细胞病毒感染的诊断和治疗方法。这些研究表明，该CMV RPE细胞模型系统可用于评估病毒复制和反义寡核苷酸抗病毒治疗的有效性。人类VZV感染眼睛可导致角膜炎、葡萄膜炎、急性视网膜坏死或进行性视网膜外坏死。然而，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VZV的豚鼠会导致慢性葡萄膜炎，包括眼后段的单个核细胞渗出。我们利用这一模型对VZV疫苗进行了评估，发现VZV gE和Gi重组蛋白免疫可诱导强烈的体液和细胞反应，加速VZV DNA的清除，并可能中和眼睛内的病毒。研究正在继续建立VZV角膜炎的体内模型。我们已经开发了使用聚合酶链式反应分析检测疱疹病毒的分子诊断方法。这些检测方法被用来区分感染性和免疫性眼球后段炎症。

Our studies of various virologic and immunopathologic processesthat 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 eye and (5) Toxoplasma gondii infections of the retina.We have established a model system for studying retinal degenerative diseases, experimental coronavirus retinopathy (ECR). 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 evaluated the role of cytokines. Analysis of cytokine levels within the eye, revealed that IL-1, Il-6, TNF-a and IFN-g were elevated in ECR. IFN-g and granule mediated cytotoxicity by cytotoxic lymphocytes (CL) play a critical role in limiting viral spread and may contribute of immunopathology. CL were analyzed by immunocytochemical staining for CD8 T cells and by RT-PCR assays for the CL cytotoxic granule, granzyme B. Sequenctial anaylsis of IFN-g and granzyme B gene expression within the retina demonstrated that these genes are up-regulated at the time of clearance of infectious virus from the retina. These studies indicate that cytotoxic T cells and T cell products (IFN-g and granzyme B) and present within the retina and contribute to retinal viral clearance. 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. 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. Human VZV infections of the eye can result in keratitis, uveitis, acute retinal necrosis or progressive outer retinal necrosis. 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. We have utilized this model to evaluate VZV vaccines and 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. Studies are continuing to establish an in vivo model for VZV keratitis. We have developed molecular diagnostic methods using PCR analysis to detect herpesviruses. These assays are being used to distinguish between infectious and immunopathogenic posterior segment intraocular inflammation.