Regulation of Enteric Virus Secretory Diarrhea

腹泻是全世界死亡和疾病的主要原因， 5岁以下儿童。轮状病毒（RV）引起约70%的病毒性腹泻，即使使用两种疫苗， 每年杀死21.5万名儿童RV腹泻主要是由于过量的Cl-分泌导致体液流失，但 负责RV诱导的液体分泌的细胞途径尚未明确，这限制了开发 并测试潜在的抗疟疾药物，可用于治疗儿童时，疫苗的保护， 不足氯离子（Cl-）分泌通道的激活受细胞内钙离子（Ca 2+）和环 这些信号反过来激活Ca 2+激活的Cl-通道（CaCC），例如 Anoctamin（Ano）家族和cAMP激活的Cl-通道（cAMP-CC），如囊性纤维化 跨膜调节因子（CFTR）。RV腹泻是由于Ca 2+升高导致Cl-分泌的典型例子 因为RV感染的一个标志是由RV非结构蛋白诱导的Ca 2+信号传导增加， 蛋白4（NSP 4）。Ano通道是肠道中主要的CaCC;然而，负责 RV腹泻尚未确定。此外，NSP 4被认为是RV诱导的Ca 2+的主要引发剂 信号;然而，NSP 4衍生的信号引起Cl-通道激活和流体的机制 分泌尚未充分研究。在目标1中，我们将确定负责RV的Cl-通道 使用体外和体内方法。关于不同Cl-的表达知之甚少。 在整个人类肠道，特别是儿童的通道，我们将使用人类肠类肠（HIE）， 确定关键Cl-通道及其调节蛋白的基因表达。接下来，我们将测量 使用荧光猝灭试验测定RV激活单个Cl-通道的能力，并确定RV是否 这些通道有助于液体分泌，使用肠样肿胀测定（ESA），在特异性 氯离子通道阻断剂。最后，我们将确定Ano 1或CFTR的抑制剂是否会减轻腹泻， 因此，肠道特异性删除这些通道是否也能减少腹泻。在目标2中，我们将确定 RV NSP 4是否通过激活传感器基质相互作用分子来激活Ca 2+和cAMP 1（STIM 1）。STIM 1激活可通过钙库操纵的钙离子进入（SOCE）产生钙信号 cAMP信号通过储存操纵的cAMP信号传导（SOcAMPs）途径。我们将 使用肠细胞系和表达荧光Ca 2+和cAMP生物传感器的HIE的活细胞成像， 确定RV和NSP 4是否激活SOCE和SOcAMP两者，并使用ESA来确定RV和NSP 4是否激活SOCE和SOcAMP两者。 由NSP 4产生的Ca 2 +/cAMP信号引起液体分泌。最后，我们将确定是否阻断剂 SOCE或SOcAMP将在体内减弱RV腹泻。我们的研究结果将产生关键的见解， 病毒性腹泻的机制和新的生物传感器HIE模型系统，可应用于未来的研究 肠道病毒，如诺如病毒和星状病毒。

Diarrheal is a leading cause of death and illness throughout the world and causes 560 million deaths of children under 5 years old. Rotavirus (RV) causes about 70% of viral diarrheas and even with two vaccines kills 215,000 children yearly. RV diarrhea is mainly due to excess Cl- secretion that drives fluid loss, but the cellular pathways responsible for RV-induced fluid secretion are not well defined, which limits efforts to develop and testing of potential anti-diarrheal drugs that can be used to treat children when protection by vaccines is insufficient. Activation of chloride (Cl-) secretory channels is regulated by intracellular calcium (Ca2+) and cyclic nucleotide (cAMP) signals These signals in turn activate Ca2+-activated Cl- channels (CaCC), such as anoctamin (Ano) family, and cAMP-activated Cl- channels (cAMP-CC), such as the cystic fibrosis transmembrane regulator (CFTR). RV diarrhea is the classic example of Cl- secretion due to elevated Ca2+ because a hallmark of RV infection is increased Ca2+ signaling, which is induced by the RV nonstructural protein 4 (NSP4). Ano channels are the prominent CaCCs in the gut; however, the Cl- channels responsible for RV diarrhea have yet to be identified. Additionally, NSP4 is considered the primary initiator of RV-induced Ca2+ signals; however, the mechanisms by which NSP4-derived signals cause Cl- channel activation and fluid secretion have not been adequately explored. In Aim 1, we will identify the Cl- channels responsible for RV diarrhea using both in vitro and in vivo approaches. Little is known about the expression of different Cl- channels throughout the human intestine, particularly children, we will use human intestinal enteroids (HIEs) to determine gene expression of the key Cl- channels and their regulatory proteins. Next, we will measure the ability of RV to activate individual Cl- channels using a fluorescence quench assay and determine whether those channels contribute to fluid secretion using the enteroid swelling assay (ESA) in the presence of specific Cl- channel blockers. Lastly, we will determine whether inhibitors of Ano1 or CFTR will attenuate diarrhea and if so whether intestinal specific deletion of those channels also reduces diarrhea. In Aim 2, we will determine whether RV NSP4 activates both Ca2+ and cAMP through activation of the sensor stromal interacting molecule 1 (STIM1). STIM1 activation can generate Ca2+ signals through the store-operated Ca2+ entry (SOCE) pathway, as well as cAMP signals through the store-operated cAMP signaling (SOcAMPs) pathway. We will use live cell imaging of intestinal cell lines and HIEs expressing fluorescent Ca2+ and cAMP biosensors to determine whether RV and NSP4 activate both SOCE and SOcAMPs and use the ESA to determine whether the Ca2+/cAMP signals generated by NSP4 cause fluid secretion. Lastly, we will determine whether blockers of SOCE or SOcAMPs will attenuate RV diarrhea in vivo. Our results will generate critical insights into the mechanisms of viral diarrhea and new biosensor HIE model systems that can be applied to future studies enteric virus, such as noroviruses and astroviruses.