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五个方面：(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)眼部水痘-带状疱疹病毒（VZV）感染；(5)视网膜弓形虫感染。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（ECR）。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中IL-1、Il-6、TNF-a和IFN-g升高。IFN-g和细胞毒性淋巴细胞（CL）颗粒介导的细胞毒性在限制病毒传播中起关键作用，并可能参与免疫病理。通过CD8 T细胞的免疫细胞化学染色和RT-PCR检测CL细胞毒性颗粒，颗粒酶B。对视网膜内IFN-g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，CMV复制和潜伏期的细胞培养模型可能为替代治疗方式的基本原理提供见解。为了了解巨细胞病毒在视网膜组织中的趋向性，我们扩展了在HRPE中巨细胞病毒复制的原始研究。这些数据表明，HRPE细胞对HCMV感染的调控在病毒进入水平和病毒基因组转录水平上都与人成纤维细胞不同。HRPE细胞中CMV与宿主细胞相互作用的差异可能有助于CMV在眼内的激活、复制和扩散。在单独的一系列研究中，我们评估了巨细胞病毒感染的诊断和治疗方法。这些研究证明了这种CMV RPE细胞模型系统在评估病毒复制和反义寡核苷酸抗病毒治疗效果方面的实用性。人类眼部VZV感染可导致角膜炎、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尚不存在。我们已经证明，在豚鼠玻璃体内接种VZV会导致慢性葡萄膜炎，其中包括眼后段的单核细胞浸润。我们利用该模型对VZV疫苗进行了评价，发现用VZV gE和gI重组蛋白免疫诱导了有效的体液和细胞反应，加速了VZV DNA的清除，并可能在眼内中和病毒。研究正在继续建立VZV角膜炎的体内模型。我们已经开发了分子诊断方法，利用PCR分析检测疱疹病毒。这些检测被用来区分感染性和免疫致病性后段眼内炎症。

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.