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过程的研究包括四个方面：(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)人类巨细胞病毒(CMV)感染的分子诊断和发病机制；(4)眼睛的疱疹病毒感染。我们已经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(EcoR)。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，我们取得了以下重要发现。由于肿瘤坏死因子-α在免疫介导的过程中起着关键作用，我们研究了视网膜变性易感的BALB/c小鼠和变性抵抗的CD-1小鼠在疾病过程中肿瘤坏死因子-α/肿瘤坏死因子-α受体和下游信号分子一氧化氮(NO)的水平。玻璃体内注射冠状病毒后，小鼠视网膜内检测到较高水平的肿瘤坏死因子-α，而血清中肿瘤坏死因子-α和肿瘤坏死因子受体1蛋白的浓度显著升高，分别为p&lt；0.005和p&lt；0.0005，但对CD-1小鼠无影响。诱导型一氧化氮合酶基因在BALB/c小鼠中最初高表达，但在感染急性期下降，而在CD-1小鼠中表达增加。这些趋势归因于菌株之间单核细胞肿瘤坏死因子受体2的释放(p&lt；0.0005)的差异，因为sTNFR2显著降低了一氧化氮的产生水平(p&lt；0.01)。这些研究表明，病毒感染后的视网膜变性与肿瘤坏死因子-a和肿瘤坏死因子受体的释放增加以及一氧化氮的下调有关。此外，他们还表明，这些分子参与了免疫反应的变化，导致了视网膜变性易感小鼠的自身免疫反应。在动物模型系统的基础上，我们还启动了评估人类视网膜退行性疾病的研究。通过免疫细胞化学染色和免疫印迹分析，对不明原因视网膜病变患者进行自身抗体检测。我们已经确认了三名锥杆变性患者，其针对神经节细胞和内核层的抗体效价很高。在已知遗传性视网膜变性患者、葡萄膜炎患者或正常人的血清中，没有检测到这些水平的抗视网膜反应。用1例视锥视杆细胞变性患者的血清，从视网膜cDNA文库中鉴定出LEDGF和ATR-X两种抗原。3例视锥-视杆细胞变性患者的血清均显示对LEDGF的反应性。识别导致视网膜细胞功能和活性改变的特异性抗视网膜抗体可能为视网膜细胞功能障碍的机制提供新的见解。 角膜疱疹感染引起强烈的炎症反应，最终导致威胁视力的间质角膜炎，这是潜伏病毒频繁重新激活的后遗症。疱疹间质角膜炎的病因学被认为是一种Th1细胞因子介导的异常免疫病理。我们评估了HSV在人角膜上皮细胞和角膜成纤维细胞中的感染情况。我们发现，单纯疱疹病毒DNA导致TLR-3和？9基因表达增强，IL-6释放增加。由于病毒通常以中和的病毒免疫复合体的形式存在，因此评估了这些免疫复合体与TLRs相互作用并触发IL-6产生的能力。我们发现单纯疱疹病毒-抗-单纯疱疹病毒免疫球蛋白复合体诱导IL-6的能力与单纯疱疹病毒DNA相当。这些现象是通过增强TLR-3和？9基因的表达来实现的。这些研究表明，在最初的急性病毒感染消退很长一段时间后，沉积在角膜基质中的病毒DNA和/或免疫复合体可能会通过TLR3和TLR9过度释放细胞因子，继续引发基质炎症。 巨细胞病毒(CMV)视网膜炎的特征是视网膜细胞功能改变和宿主对病毒复制的反应。我们评价了环氧合酶-2(COX-2)和前列腺素(PGE)对CMV感染的人视网膜色素上皮(RPE)细胞的诱导作用，并确定了它们对病毒复制的影响。在巨细胞病毒视网膜炎患者的视网膜组织切片中检测到CMV即刻早期(IE)蛋白和环氧合酶-2(COX-2)蛋白。培养的人RPE细胞感染CMV后，COX-2mRNA和蛋白均被诱导表达。巨细胞病毒感染RPE细胞可诱导核转录因子-kB由胞浆移位至胞核。人视网膜色素上皮细胞感染巨细胞病毒后，前列腺素E_1和前列腺素E_2显著升高(P<0.001)。反义寡核苷酸抑制CMV IE基因可抑制COX-2的mRNA表达和COX-2、PGE2的蛋白合成。PGE促进了CMV空斑的形成，实时荧光定量聚合酶链式反应分析表明，PGE处理显著增加了CMV的DNA拷贝数。这些研究表明，当CMV在人RPE细胞内复制时，COX-2诱导通过PGE途径增加病毒复制。在视网膜CMV感染过程中，COX-2和PGE的诱导可能会增强病毒的复制，改变视网膜的各种生理反应。

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. Since TNF-a plays a crucial role in immune-mediated processes we evaluated the levels of TNF-a/TNF-a receptors and the downstream signaling molecule nitric oxide (NO) during the course of disease in both retinal degeneration susceptible BALB/c and degeneration resistant CD-1 mice. Following intravitreal injection with the coronavirus, mouse hepatitis virus (MHV), TNF-a mRNA was detected at higher levels within the retinas, and concentrations of TNF-a and sTNFR1 proteins were significantly increased, p<0.005 and p< 0.0005 respectively, within the serum of BALB/c but not CD-1 mice. Gene expression of iNOS while initially high in BALB/c mice decreased during the acute phase of infection, while it increased in CD-1 mice. These trends are attributable to differences in monocyte TNFR2 release (p < 0.0005) between the strains since sTNFR2 significantly decreased (p < 0.01) levels of NO production. These studies demonstrate that retinal degeneration following viral infection is associated with increased release of TNF-a and TNF receptors combined with a down-regulation of NO. Furthermore they suggest that these molecules are involved in alterations in immune response leading to the autoimmune reactivity seen in retinal degeneration susceptible mice. 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.