PATHOGENESIS- HUMAN CALICIVIRUSES IN GNOTOBIOTIC ANIMALS

说明(改编自应用程序) 人类肠道杯状病毒(HuCV)是引起急性疫情的主要原因 胃肠炎，占食源性肺炎病例的67% 在美国每年都有人生病。尽管付出了相当大的努力，但主要集中在 诺沃克HuCV原型，试图在细胞内培养挑剔的HuCV 培养或开发动物疾病模型的努力都失败了。我们缺少的是 人类疱疹病毒致病机制、复制、血清型和宿主免疫的认识 阻碍制定预防人类免疫缺陷病毒感染的战略。基于序列 分析表明，人乳头瘤病毒存在3个基因组群：诺瓦克样病毒(NLV)I、II和 札幌类病毒(SLV)。最近我们和其他人发现肠道 猪源杯状病毒(PEC)和牛源杯状病毒(BEC)在基因上更接近 与HuCV相关的病毒比与其他动物杯状病毒更近。此外，猪的粪便 小牛SLV、NLV II和NLV IRNA分别为阳性 HuCV的引物，暗示了HuCV或 可传播给人类的人畜共患病菌株。因此，我们的目标是发展动物 研究人类和动物杯状病毒比较发病机制的模型， 以NLV I型毒株和NLV II、SLV毒株为靶点的Gn犊牛 在Gn猪身上。我们将探索的新策略包括使用血清阴性， 新生儿Gn动物宿主，替代接种途径(静脉接种 提供比口服更低的病毒剂量)和药理免疫抑制 以增强寄主的敏感性。待评估的参数包括寄主年龄、 临床症状、病毒血症、病毒脱落、血清转换和抗体影响 再次暴露，器官和细胞类型被感染，包括抗原和病变 分发。分析方法包括电子显微镜、逆转录聚合酶链式反应和 重组VLP衣壳免疫荧光法检测病毒 抗体检测和组织病理学检查用于病变检查。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感染性的克隆 减毒的PEC并对其进行系统突变以反映 然后检测突变株对Gn猪的致病性。我们的发现 应该为人类和人类的相对致病机制提供新的信息 动物杯状病毒，其毒力的潜在遗传基础和 为今后宿主免疫和预防策略的研究奠定基础。

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.