Role of the innate immune response in canine distemper virus neuropathogenesis

犬瘟热病毒(CDV)在多种食肉动物中引起严重的全身性疾病。此外，多达30%的狗经历了中枢神经系统(CNS)的受累，这使得CDV非常适合描述MorbilliVirus诱导的神经病理机制。在中枢神经系统，CDV引起急性和慢性脱髓鞘损害，部分是由免疫病理引起的。由于CDV V蛋白在抑制抗病毒反应中起着至关重要的作用，我假设CDV相关的炎性中枢神经系统损害和随后的脱髓鞘是由于在急性疾病阶段先天免疫反应对感染的控制不足造成的。为了验证这一假设，我提出了一项具有两个特定目标的研究计划：(A)评估CDV在不同类型的原代芥状细胞和犬细胞中抑制干扰素(干扰素)信号级联的能力；以及(B)表征CDV在不同类型的原代芥菜和犬细胞中的扩散、靶细胞、以及野生型和V缺失病毒在芥子虫器官型脑片培养中的局部先天免疫应答。对于(A)，我将测定不同原代芥虫和犬中枢神经系统细胞类型、原代上皮细胞和外周血单核细胞的复制效率。我还将使用VSV生物测定法测量I型干扰素的产生，以调查复制效率与先天免疫激活程度之间的相关性。对于(B)，我将首先通过免疫组织化学方法比较两种病毒的传播细胞和靶细胞，并通过VSV生物检测来评估先天免疫激活，通过实时定量RT-PCR和Western印迹分析来评估细胞因子的表达水平。综上所述，这项工作将促进我们对宿主反应对麻疹病毒相关神经并发症的贡献的理解，并将为开发有针对性的预防性治疗奠定基础。

Canine distemper virus (CDV) causes a severe systemic disease in a broad range of carnivores. In addition, up to 30% of dogs experience involvement of the central nervous system (CNS), making CDV well suited for the characterization of Morbillivirus-induced neuropathogenetic mechanisms. In the CNS, CDV causes acute and chronic demyelinating lesions, which are partially of immunopathogenic origin. Since the CDV V-protein plays an essential role in the inhibition of antiviral responses, I hypothesize that CDV-associated inflammatory CNS lesions and subsequent demyelination result from an insufficient infection control by the innate immune response during the acute disease phase.To test this hypothesis, I am proposing a research plan with two specific aims:(a) To assess the ability of CDV to inhibit the interferon (IFN) signaling cascade in different primary mustelid and canine cell types, and (b) to characterize the dissemination, target cells, and the local innate immune response of wild type and V-deleted viruses in mustelid organotypic brain slice cultures.For (a) I will determine the replication efficiency in different primary mustelid and canine CNS cell types, primary epithelial cells, and peripheral blood mononuclear cells. I will also measure the type I IFN production using a VSV-bioassay to investigate a correlation between replication efficiency with the extent of innate immune activation. For (b) I will first compare the spread and target cells of the two viruses by immunohistochemistry, and assess the innate immune activation by VSV-bioassay and cytokine expression levels using real-time RT-PCR and Western blot analysis. The cytokine-expressing cells will then be identified by double-staining of tissue sections.Summarized, the proposed work will advance our understanding of the host response contribution to morbillivirus-associated neurologic complications and will constitute the basis for the development of a targeted preventative therapy.