Using a novel neonatal mouse model to determine mechanisms of norovirus-induced disease

项目摘要/摘要 诺沃克病毒是全世界儿童严重腹泻的主要原因，也是急性腹泻的主要原因 各年龄组均有胃肠炎。目前还没有批准的诺沃克病毒疫苗或靶向治疗药物 感染，对胃肠炎症状的致病机制知之甚少。为了获得 随着对这种重要病毒的进一步了解，多年来一直使用小鼠诺沃克病毒作为模型系统 并在理解诺沃克病毒生物学方面取得了许多重大进展。然而，由于没有 感染小鼠诺如病毒的免疫活性成年小鼠的症状限制了该模型的适用性 病毒致病机制的描述。我们最近发现，基因野生型新生小鼠 感染小鼠诺如病毒后出现急性自行解决的腹泻，其病程与人类相似 诺沃克病毒感染。这一新的诺沃克病毒病小动物模型代表了在 诺沃克病毒领域，因为它将能够全面描述病毒疾病的机制，并最终服务于 作为测试体内抗病毒化合物疗效的平台。在开发这种新模型时，我们观察到 基因相似的小鼠诺如病毒株之间疾病严重程度的差异使病毒得以识别 诺沃克病毒引起疾病的决定因素。这项提议的目的是阐明细胞 利用我们新的症状Small研究诺如病毒腹泻的嗜性和致病机制 动物模型。在具体目标1中，我将检验淋巴细胞感染对诺沃克病毒至关重要的假设- 诱发性腹泻。这一假说得到了我们的体外研究结果的有力支持，这些研究结果表明，尽管毒性很强， 而减毒株在巨噬细胞中的复制情况相似，只有强毒株在淋巴细胞中复制。 此外，在体外消除淋巴细胞感染的突变正在减弱成年干扰素缺陷小鼠的病情。在……里面 具体目标2，我将测试病毒诱导的促炎细胞因子在TRAIL中引起干扰的假设 连接维持肠道上皮屏障，从而导致肠道通透性增加 还有拉肚子。这一假设是基于我们的观察结果，即一种毒力强的小鼠诺沃克病毒株可以诱导 尽管病毒水平相当，但致炎细胞因子的表达明显高于减毒株 复制；以及已确立的促炎细胞因子破坏紧密连接的能力。总体而言， 这项建议中描述的研究将测试我们的模型，即诺沃克病毒感染肠道免疫细胞诱导 一种免疫病理的宿主反应，通过对肠道上皮屏障的影响而导致腹泻。

Project Summary/Abstract Norovirus is the leading cause of severe childhood diarrhea around the world and a major cause of acute gastroenteritis in all age groups. There are no currently approved vaccines or targeted therapeutics for norovirus infection and very little is known about the pathogenic mechanisms underlying gastroenteritis symptoms. To gain further understanding of this important virus, murine norovirus has been used as a model system for many years and has led to many significant advances in understanding norovirus biology. However, the absence of symptoms in immunocompetent adult mice infected with murine norovirus limits the applicability of this model to delineation of viral mechanisms of disease. We recently discovered that genetically wild-type neonatal mice develop acute, self-resolving diarrhea when infected with murine norovirus, a disease course that mirrors human norovirus infection. This novel small animal model of norovirus disease represents a major advance in the norovirus field since it will enable a complete characterization of viral disease mechanisms and ultimately serve as a platform to test the efficacy of antiviral compounds in vivo. While developing this new model, we observed differences in disease severity between genetically similar murine norovirus strains enabling identification of viral determinants of norovirus-induced disease. The objectives of this proposal are to elucidate the cellular tropism and pathogenic mechanisms underlying norovirus diarrhea using our novel symptomatic small animal model. In Specific Aim 1, I will test the hypothesis that infection of lymphocytes is critical to norovirus- induced diarrhea. This hypothesis is strongly supported by our in vitro findings revealing that, although virulent and attenuated strains replicate comparably in macrophages, only the virulent strain replicates in lymphocytes. Moreover, mutations abolishing lymphocyte infection in vitro are attenuating in adult interferon-deficient mice. In Specific Aim 2, I will test the hypothesis that virus-induced proinflammatory cytokines cause disruptions in tight junctions maintaining the intestinal epithelial barrier, consequently leading to increased intestinal permeability and diarrhea. This hypothesis is based on our observation that a virulent murine norovirus strain induces significantly more proinflammatory cytokine expression than attenuated strains despite comparable levels of viral replication; and on the well-established ability of proinflammatory cytokines to disrupt tight junctions. Overall, the studies described in this proposal will test our model that norovirus infection of intestinal immune cells induces a host response that is immunopathologic and leads to diarrhea via effects on the intestinal epithelial barrier.