VIRUS INFECTIONS IN THE EYE

我们对病毒和寄生虫在眼部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）病毒诱导的视网膜变性过程;（2）病毒在人类疾病中的可能作用;（3）人类巨细胞病毒（CMV）感染的分子诊断和发病机制;（4）水痘-带状疱疹病毒（VZV）眼部感染;（5）弓形虫视网膜感染。该病毒能够在存在轻度视网膜血管炎症的情况下诱导急性感染。最初的视网膜损伤之后是感染性病毒的清除和进行性视网膜变性。这是第一个证明病毒诱导的变性、病毒持续存在、对病毒诱导的组织损伤的遗传易感性和病毒触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变性疾病的病理生理机制，并确定相关基因。 在过去的一年中，我们评估了细胞因子的作用。 眼内细胞因子水平的分析显示，IL-1、IL-6、TNF-α和IFN-γ在ECR中升高。 IFN-γ和颗粒介导的细胞毒性淋巴细胞（CL）的细胞毒性发挥关键作用，限制病毒传播，并可能有助于免疫病理学。通过CD 8 T细胞的免疫细胞化学染色和CL细胞毒性颗粒、颗粒酶B的RT-PCR测定来分析CL。视网膜内IFN-γ和颗粒酶B基因表达的序列分析表明，这些基因在感染性病毒从视网膜清除时上调。这些研究表明，细胞毒性T细胞和T细胞产物（IFN-g和颗粒酶B）存在于视网膜内并有助于视网膜病毒清除。人CMV是一种疱疹病毒，是先天性感染儿童和免疫功能低下个体失明的主要原因。 研究人类巨细胞病毒潜伏期是困难的，因此，巨细胞病毒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依据。 为了了解视网膜组织嗜性CMV，我们已经扩大了我们原来的研究CMV复制HRPE。 这些数据表明，HCMV感染HRPE细胞的调节在病毒进入和病毒基因组转录水平上均不同于人成纤维细胞。 HRPE细胞中CMV -宿主细胞相互作用的这些差异可能有助于CMV在眼内的活化、复制和传播。 在一系列独立的研究中，我们评估了CMV感染的诊断和治疗方法。这些研究证明了该CMV RPE细胞模型系统用于评估病毒复制和使用反义寡核苷酸的抗病毒治疗的功效的效用。人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外视网膜坏死。 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的豚鼠的结果在慢性葡萄膜炎组成的单核细胞浸润在眼睛的后段。 我们已经利用这个模型来评估VZV疫苗，并发现用VZV gE和gI重组蛋白免疫诱导了有效的体液和细胞应答，加速了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PCR分析来检测疱疹病毒的分子诊断方法。 这些试验被用于区分感染性和免疫病理性眼内后段炎症。

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.