PATHOGENESIS- HUMAN CALICIVIRUSES IN GNOTOBIOTIC ANIMALS

描述（改编自应用程序） 人类肠道杯状病毒（HuCV）是急性流行病的主要原因 胃肠炎，占食源性疾病病例的 67% 每年在美国生病。尽管付出了巨大的努力，但主要集中在 Norwalk HuCV原型，尝试在细胞中培养挑剔的HuCV 培养或建立动物疾病模型都失败了。我们缺乏 了解 HuCV 发病机制、复制、血清型和宿主免疫 阻碍制定预防 HuCV 感染的策略。基于顺序 分析发现，HuCV 存在 3 个基因组：诺沃克样病毒 (NLV) I 和 II 以及 札幌样病毒（SLV）。最近我们和其他人发现肠溶 来自猪 (PEC) 和小牛 (BEC) 的杯状病毒在遗传上更为接近 与 HuCV 的相关性高于与其他动物杯状病毒的相关性。此外，猪的粪便 和小牛分别使用 SLV 和 NLV II 以及 NLV I RNA 呈阳性 HuCV 的引物，表明 HuCV 动物宿主的可能性或 可传播给人类的人畜共患病菌株。因此我们的目标是开发动物 研究人类和动物杯状病毒的比较发病机制的模型， 靶向限生 (Gn) 小牛中的 NLV I 菌株以及 NLV II 和 SLV 菌株 在 Gn 猪中。我们将探索的新策略包括使用血清阴性、 新生 Gn 动物宿主，替代接种途径（静脉注射 与口服相比，提供较低的病毒剂量）和药物免疫抑制 以增强宿主的敏感性。要评估的参数包括宿主年龄、 临床症状、病毒血症、病毒脱落、血清转化和抗体对患者的影响 再次暴露，器官和细胞类型被感染，包括抗原和病变 分布。分析方法包括电子显微镜、RT PCR 和 免疫荧光用于病毒检测，ELISA（重组VLP衣壳）用于 抗体检测和组织病理学用于病变检查。最后我们的一对 毒力和减毒、细胞适应的 PEC/Cowden 及其知识 序列差异（总共 7 个氨基酸差异）使我们能够探索 PEC毒力的分子基础。我们计划构建一个具有传染性的克隆 减弱的 PEC 并系统地对其进行突变以反映 强毒 PEC，然后测试突变体在 Gn 猪中的致病性。我们的发现 应提供关于人类和人类的比较发病机制的新信息 动物杯状病毒，其毒力的潜在遗传基础和 为未来宿主免疫和预防策略的研究奠定了基础。

DESCRIPTION (adapted from the application) Human enteric caliciviruses (HuCV) are the leading cause of acute epidemic gastroenteritis and they account for 67 percent of the cases of foodborne illness in the U.S. annually. Despite considerable efforts, focused mainly on the prototype Norwalk HuCV, attempts to cultivate the fastidious HuCV in cell culture or to develop an animal disease model have failed. Our lack of understanding of HuCV pathogenesis, replication, serotypes and host immunity impedes development of strategies to prevent HuCV infections. Based on sequence analysis, 3 genogroups of HuCV exist: Norwalk-like viruses (NLV) I and II and Sapporo-like viruses (SLV). Recently we and others found that enteric caliciviruses from pigs (PEC) and calves (BEC) are genetically more closely related to HuCV than to other animal caliciviruses. Moreover, feces from pigs and calves were positive for SLV and NLV II, and NLV I RNA, respectively using primers for HuCV, suggesting the possibility of animal reservoirs for HuCV or zoonotic strains transmissible to humans. Thus our goal is to develop animal models to study the comparative pathogenesis of human and animal caliciviruses, targeting NLV I strains in gnotobiotic (Gn) calves and NLV II and SLV strains in Gn pigs. New strategies we will explore include the use of seronegative, neonatal Gn animal hosts, alternative inoculation routes (intravenous for delivery of lower virus doses versus oral) and pharmacologic immunosuppression to enhance host susceptibility. Parameters to be assessed include host age, clinical signs, viremia, virus shedding, seroconversion and antibody impact on re-exposure, and organs and cell types infected including antigen and lesion distribution. Methods of analysis include electron microscopy, RT PCR and immunofluorescence for virus detection, ELISA (recombinant VLP capsid) for antibody detection and histopathology for lesion examination. Finally our pair of virulent and attenuated, cell-adapted PEC/Cowden and knowledge of their sequence differences (7 total amino acid differences) allows us to explore the molecular basis for PEC virulence. We plan to construct an infectious clone of the attenuated PEC and systematically mutate it to mirror the sequence of the virulent PEC, then test the mutants for pathogenicity in Gn pigs. Our findings should provide new information on the comparative pathogenesis of human and animal caliciviruses, the potential genetic basis for their virulence and a foundation for future studies of host immunity and preventive strategies.