PATHOGENESIS- HUMAN CALICIVIRUSES IN GNOTOBIOTIC ANIMALS

描述（改编自应用程序） 人肠道杯状病毒（Human intestinal calicivirus，HuCV）是引起急性流行病的主要原因 肠胃炎，他们占百分之六十七的情况下，食源性 每年在美国发病。尽管作出了相当大的努力， Norwalk HuCV的原型，试图在细胞中培养挑剔的HuCV， 培养或开发动物疾病模型都失败了。我们缺乏 了解HuCV的发病机制、复制、血清型和宿主免疫 阻碍了预防HuCV感染的策略的发展。基于序列 分析，存在3个HuCV基因组：诺瓦克样病毒（NLV）I和II， 札幌样病毒（SLV）。最近我们和其他人发现， 来自猪（PEC）和小牛（BEC）的杯状病毒在遗传上更接近 与其他动物杯状病毒相比，与HuCV的相关性更高。此外，猪的粪便 和小牛分别为SLV和NLV II和NLV I RNA阳性， HuCV的引物，表明HuCV的动物储库的可能性， 人畜共患病菌株可传染给人类。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， 靶向无菌（Gn）小牛中的NLV I毒株以及NLV II和SLV毒株 在猪中。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， 递送较低的病毒剂量（相对于口服）和药理学免疫抑制 增强宿主易感性待评估的参数包括宿主年龄， 临床体征、病毒血症、病毒脱落、血清转化和抗体对 再次暴露，以及器官和细胞类型感染，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 抗体检测和病变组织病理学检查。最后我们的搭档 毒力和减毒，细胞适应PEC/考登和他们的知识， 序列差异（7个氨基酸差异）使我们能够探索 PEC毒力的分子基础。我们计划构建一个感染性克隆 减弱的PEC，并系统地使其突变，以反映 致病性PEC，然后在Gn猪中测试突变体的致病性。我们的研究结果 应该提供新的信息比较发病机制的人类和 动物杯状病毒，其毒力的潜在遗传基础和 为今后研究宿主免疫和预防策略奠定基础。

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.