单纯疱疹病毒性角膜炎树鼩模型的建立及发病机理研究

Herpes Simplex Virus type I (HSV-1) infection causes inflammation in the cornea known as Herpes simplex virus keratitis (HSK), a common but serious corneal disease. Due to the lack of an animal model that closely mimic human HSK, very little is known on the extent of viral infection within each anatomical structures of the eye, and how the pathological, immunological responses unfold. More importantly, it is not entirely clear whether the reactivating virus come from the trigeminal ganglia, within the cornea or eyes deep tissue. Our recent published studies of HSV-1 infection in the tree shrew trigeminal ganglia and CNS suggested that tree shrews infection is more similar to humans than that of the rodent models. Tree shrew is evolutionarily closer to primates than rodent. Interestingly, the anatomic structures of the tree shrew eye are similar to that in humans. For these reasons, we studied HSV-1 infection in tree shrew cornea and found that HSK symptom in tree shrews is also very closely resembles that of the human HSK. In addition, we also observed that HSV-1 latency transcript LAT is expressed in neuronal cells in the ciliary ganglia, suggesting that HSV-1 may latently or persistently these cells. In this application, we propose to use tree shrew corneal infection as a model of human HSK to further investigate this disease. First, we will infect tree shrew eyes by HSV-1 to induce HSK by corneal scratching to establish tree shrew HSK model. Next, we will characterize HSV-1 lytic infection, latent infection and reactivation in tree shrews eyes. Then we will test whether HSV-1 latently or persistently infect eye tissues, particularly neuronal cells in the cornea, retina and other eye tissues. If funded, the proposed study could establish a novel non-human primate HSK model, which will benefit mechanistic studies, drug development and therapeutic intervention of this disease.

单纯疱疹病毒性角膜炎(Herpes simplex keratitis, HSK)，为常见且严重的角膜病。由于潜伏感染的疱疹病毒可以重激活，HSK易复发，不能根治。但目前人们对病毒潜伏的位点还有很大争议，也没有更接近于人的灵长类疱疹病毒性角膜炎模型报道。我们发现树鼩较啮齿类模型能更好地模拟人类HSK症状，病毒在睫状神经节有潜伏。为进一步研究疱疹病毒感染树鼩角膜及眼睛深部组织的病理变化，探索病毒潜伏和重激活的机制，本研究计划：1）用HSV-1通过眼膜接种途径感染树鼩，建立和鉴定HSK树鼩模型，模拟疱疹病毒性角膜炎的发生，发展及复发过程。2）利用免疫组化、原位杂交和体外共培养等方法，检测病毒在树鼩角膜、视网膜及眼睛深部组织的潜伏和重激活位点，解析疱疹病毒性角膜炎反复发病的机理。本研究将为疱疹病毒性角膜炎的临床治疗提供有用的基础数据和理想的筛药模型。

单纯疱疹病毒HSV-1感染引起的角膜炎症称为单纯疱疹病毒性角膜炎(Herpes simplex keratitis, HSK)，为常见且严重的角膜病，居角膜病发病率和致盲原因的首位。一旦病毒再激活，HSK易复发，不能根治，也是眼科手术包括角膜移植的难题。疱疹病毒性角膜炎的复发，病毒是来源于潜伏在TG细胞内的病毒再活化，通过轴浆运输到达角膜，还是潜伏在角膜或眼睛深部组织（包括视网膜和睫状神经节）的病毒直接重激活，这些并不是很清楚。.目前，家兔和啮齿类是研究疱疹病毒性角膜炎的主要动物模型，但由于家兔和啮齿类与灵长类差距甚远，并不能完整地模拟人类疱疹病毒性角膜炎的感染情况。树鼩更接近于灵长类，树鼩眼睛的组织结构更接近于人的眼睛，我们通过眼睛接种途径用HSV-1感染树鼩进行研究发现：树鼩模型能很好地模拟人的疱疹病毒性角膜炎症状，急性感染期可观察到典型的点状，树枝状病毒性角膜炎症状。随着HSV-1的感染，导致如下病变和并发症：角膜基质瘢痕形成，角膜增厚（初发感染），角膜透明度降低，新生血管生成。在HSV-1感染的树鼩模型中，其角膜的上皮细胞和视网膜神经元细胞中可检测到HSV-1病毒蛋白。HSV-1的病毒转录子，ICP0, ICP4和LAT 在感染后3天能被检测到，峰值出现在感染后5天，两周后，ICP0和ICP4的转录表达降到一个基础水平，但LAT 的表达量有所增加，至感染后46天，LAT积累至一个很高的表达水平。原位杂交也在潜伏期树鼩眼睛中检测到潜伏相关转录子LAT的表达。有趣的是：急性感染期后，我们仍然在树鼩的眼睛检测到大量的活病毒，进一步的研究发现，潜伏感染期树鼩的角膜和睫状神经节中存在持续感染的HSV-1病毒，预示着树鼩有可能会复发疱疹病毒性角膜炎。这些研究结果为疱疹病毒性角膜炎树鼩模型的可行性提供了充分的依据，树鼩模型将成为疱疹病毒性角膜炎疾病机理研究的有用模型。