诺如病毒的功能性共受体鉴定及石榴皮有效部位的抗病毒机制研究

Noroviruses (NoVs, belong to Caliciviridae) are the most common cause of non-bacterial gastroenteritis outbreak.NoVs are difficult to study owing to lack of a cell culture and efficient small animal model. Currently there is no antiviral, vaccine or other effective intervention against NoVs. NoVs just attach to the cell membrane, and high-copy virus replication still cannot be achieved when cultured in differentiated Caco-2 cell line with stable HBGA-H antigen expression. In recent years, some advancement was achieved on co-receptor of caliciviruses, and tight junction proteins were discovered and identified as the functional co-receptor for caliciviruses. In this project, we hypothesize that tight junction proteins are the functional co-receptor for NoV. Since a monkey calicivirus (Tulane virus, TV) was successfully cultured and purified in MK2 cell, we will try to use this TV cell culture system as a breaking point. To identify the functional co-receptor of NoV, tight junction proteins, such as occludin，claudin，JAM and ZOs, will be the target; Protein function assay and receptor reconstruction technique will be used. The discovery of co-receptor of NoV is great significance to elucidate mechanism of NoV infection and provide new target of anti-NoV drug development. At the same time, we found out Pomegranate has significant blocking effect against NoV binding to HBGA receptor in our previous studies. In this project, we will use our newly developed perfect NoV surrogate model (TV cell culture system) to evaluate the anti-viral effect, active composition and mechanism of Pomegranate. This project will further provide new idea and scientific basis to development of ant-viral natural drug for NoV.

诺如病毒（NoV）不能体外培养亦没有小动物感染模型，迄今没有特异性抗NoV药物，其感染机制也不明确。基于NoV在亲嗜性Caco-2细胞中仍不能感染复制的困境，结合杯状病毒受体研究新进展和启示，本项目提出"NoV的功能性共受体是某紧密连接蛋白"假说，以猴杯状病毒（TV）正成为NoV理想的体外培养替代模型为突破口，着眼于occludin，claudin，JAM和ZO及其家族成员蛋白，采用流式细胞术、病毒铺覆蛋白结合法、空斑抑制实验和受体重建等技术，验证该假说从而鉴定出NoV的共受体。同时，在发现石榴皮具有显著阻断NoV衣壳蛋白结合人类组织血型抗原（HBGA）受体作用的基础上，大孔吸附树脂法提取分离主要有效部位，利用TV替代模型，深入评价它们的体外抗病毒效果并初步阐明其主要作用机制。这将有助于深入理解NoV的感染机制，为进一步开发抗NoV天然药物提供科学的实验依据，具有重要的理论价值和科学意义。

诺如病毒（Norovirus，NoV）是全球范围内引起成人和儿童非菌性急性胃肠炎暴发流行的重要病原体，目前已明确肠道黏膜上的人类组织血型抗原（human histo-blood group antigens，HBGAs）是NoV的病毒受体。本项目建立猴杯状病毒（TV）体外培养替代模型，开展hNoV的受体及中药石榴皮的抗病毒作用机制研究。研究结果表明：（1）神经节苷脂（gangliosides）中含有的唾液酸成分sialic acid (SA)在hNoV和TV感染中起到重要配体作用，两者在受体上具有一定的相似性；（2）与HBGA受体的广谱结合作用可能是GII.17 NoV近两年来大规模暴发流行的主要分子机制；来源于GII.4和 GII.17的血清交叉免疫反应很弱，但具有一定的交叉受体阻断作用，GII.17作为一种重要的抗原类型在未来hNoV的疫苗研制策略中应该加以重视；（4）轮状病毒（RV）作为病毒性胃肠炎的主要病原体，其感染亦与HBGA分泌型有关，RV与HBGA的结合模式可能是P[8]和P[4] RV成为主要流行株的重要机制，该研究为NoV的受体研究提供了有益的启示。（5）中药石榴皮水提液10µg/ml浓度即具有明显的直接杀病毒作用和抗病毒效果。最新研究进展表明NoV和共生菌通过细胞内吞作用感染粘膜下靶细胞（B细胞），这提示本项目提出的“NoV的功能性共受体是某紧密连接蛋白”理论假说尚存在一定的不确定性，后期仍需运用最新的NoV细胞培养模型深入研究。本项目研究已发表2区SCI论文4篇，中文核心期刊1篇。两次邀请美国辛辛那提大学儿童医学研究中心Jiang xi教授团队学术交流，并选派博士研究生1名访问研究一年，培养硕士研究生2名。