The role of gut microbiota in human norovirus infections in transplant patients

项目总结 人类诺如病毒(HuNoV)是美国急性胃肠炎的主要原因，导致4.2美元 每年的直接医疗系统成本为10亿美元。由于潜在的免疫抑制和缺乏有效的 如果接受抗病毒治疗，移植患者可能会因感染新城疫病毒而出现严重的后遗症。尽管 肠道微生物区系已被证明在临床前可增强肠道病毒的复制和致病作用。 在模型中，它在人新城疫病毒感染中的作用在很大程度上仍不清楚。在这个项目中，我们的目标是获得更大的机械性 肠道微生物区系如何调节移植患者的新城疫感染。我们的中心假设是 患有有症状的HuNov感染的移植患者将有肠道微生物组特征，显示出 促进HIE感染的特定肠道微生物区系(肠杆菌科)的丰富，以及那些 感染新城疫病毒后出现慢性症状会伴随特定肠道微生物区系的枯竭 调节宿主的先天免疫反应(1型干扰素)。在目标1中，我们将进一步定义这两个Gut 移植患者微生物群和宿主因子的差异±HuNoV感染。第一，我们将建立更大的 对成人和儿童移植患者进行队列调查，并收集纵向粪便标本。然后，我们将表演 全面的肠道微生物组图谱(元基因组鸟枪测序和细菌qPCR)以确认我们的 初步结果。我们预测，感染HuNov的移植患者的肠道将有明显的不同 与未感染的微生物区系特征相比，以及更高强度的免疫抑制和 在感染HuNoV的移植患者中，高抗生素负荷量对应于慢性腹泻。在目标2中，我们将 使用体外HIE模型确定肠杆菌科是否促进HuNoV感染。首先，我们会感染空肠 HIE与HuNov共孵育，并与多种肠杆菌科细菌共孵育。然后，我们将进行RNA 提取液和定量HuNoV RT-qPCR评价不同肠杆菌科细菌共培养的效果 SPP.关于病毒复制的研究。我们预测肠杆菌科促进HIE的HuNoV感染。如果这是 我们将调查这种现象是否只在表达HBGA的成员中观察到 肠杆菌科。在目标3中，我们将确定移植患者是否患有慢性有症状的HuNov感染 与未感染的患者相比，具有不同的系统性细胞因子特征。我们将执行大量的细胞因子 利用新的功能蛋白质组学分析平台Isoplexis分析血清样品。我们预测 患有新城疫病毒感染的慢性腹泻的移植患者将表现出类杆菌属的稀少， 导致1型干扰素耗尽，从而导致Th1免疫反应下降，增加 Th2免疫应答和Th2优势细胞因子(IL-4、IL-5、IL-10)表达增加。建议数 K23课程中的实验、培训和教学课程将为候选人(庄博士)配备独特的 使她能够作为肠道微生物区系的内科科学家过渡到独立的技能-HuNov 移植患者之间的相互作用。

PROJECT SUMMARY Human norovirus (HuNoV) is the leading cause of acute gastroenteritis in the United States, resulting in $4.2 billion in direct health system costs annually. Due to underlying immunosuppression and the lack of effective antiviral therapeutics, transplant patients may develop serious sequelae from HuNoV infections. Though bacterial gut microbiota has been shown to enhance replication and pathogenesis of enteric viruses in preclinical models, its role in HuNoV infection remains largely unknown. In this project, we aim to gain greater mechanistic insights into how gut microbiota modulate HuNoV infection in transplant patients. Our central hypothesis is that transplant patients with symptomatic HuNoV infection will have a gut microbiome signature showing an enrichment of specific gut microbiota (Enterobacteriaceae) that facilitate infection of HIEs, and those who develop chronic symptoms from HuNoV infection will have a concomitant depletion of specific gut microbiota that modulate host innate immune responses (type 1 interferons). In Aim 1, we will further define both gut microbiome and host factor differences in transplant patients ±HuNoV infections. First, we will establish a larger cohort of adult and pediatric transplant patients and collect longitudinal stool specimens. Then, we will perform comprehensive gut microbiome profiling (metagenomic shotgun sequencing and bacterial qPCR) to confirm our preliminary results. We predict that HuNoV-infected transplant patients will have significantly different gut microbiota signatures compared to uninfected counterparts, and that higher intensity of immunosuppression and high antibiotic load will correspond with chronic diarrhea in HuNoV-infected transplant patients. In Aim 2, we will determine if Enterobacteriaceae facilitate HuNoV infection using an in vitro HIE model. First, we will infect jejunal HIEs with HuNoV and co-incubate HuNoV with various Enterobacteriaceae spp. Then, we will perform RNA extractions and quantitative HuNoV RT-qPCR to evaluate the effect of co-incubating various Enterobacteriaceae spp. on viral replication. We predict that Enterobacteriaceae promotes HuNoV infection of HIEs. If this is the case, we will investigate if this is a phenomenon observed only in HBGA-expressing members of Enterobacteriaceae. In Aim 3, we will determine if transplant patients with chronic symptomatic HuNoV infections have different systemic cytokine signatures compared to uninfected counterparts. We will perform bulk cytokine analysis on serum samples utilizing Isoplexis, a novel functional proteomic profiling platform. We predict that transplant patients with chronic diarrhea from HuNoV infection will exhibit a paucity of genus Bacteroides, resulting in depletion of type 1 interferons, which then leads to a decreased Th1 immune response, increased Th2 immune response and increased expression of Th2-predominant cytokines (IL-4, IL-5, IL-10). The proposed experiments, training and didactic coursework in this K23 will equip the candidate (Dr. Chong) with unique skillsets that will enable her transition to independence as a physician scientist in gut microbiota-HuNoV interactions in transplant patients.