人源诺如病毒与生菜叶的互作机制

Human norovirus (hNoV) is an important foodborne virus. The romaine lettuce is one of the main vehicles for dissemination of hNoV. To discover the interaction mechanism between the hNoV and the romaine lettuce leaf, it is a breakthrough to investigate the specific ligands in the romaine lettuce leaf for binding to the viruses, and the key sites in the capsid protein for binding with the specific ligands. In this project, a modified mimic hNoV had been constructed by the bacterial surface display system, which was enlarged hundredfold comparing with hNoV particle. The mimic hNoV will be used as a mediator to capture and bioaccumulate the specific ligands in the romaine lettuce leaf, which binding with the capsid protein of different genotype hNoV. The specific ligands, including saccharides and proteins, will be purified and identified. The functional group of the specific ligands will be chartered by enzyme digestion and competitive enzyme linked immunosorbent assay (ELISA). The single or multiple sites mutation library of P domain in the capsid protein will be built by a commercial kit. The binding capability will be evaluated between the mutant P domain of capsid protein displayed on the surface of mimic hNoV and specific ligands in the romaine lettuce leaf. The key amino acids in the P domain will be located. The crucial domain in capsid protein will be explored, which plays an important role in specific binding between the virus and the romaine lettuce. In addition, the dynamic of interaction between the P domain of capsid and specific ligands was confirmed by surface plasmon resonance (SPR). Finally, the interaction mechanism between the foodborne virus and the romaine lettuce leaf will be summarized. Results in this project will be benefit to build up a system for prevention and control of hNoV in fruits and vegetables.

人源诺如病毒（human norovirus，hNoV）是重要的食源性病毒，生菜是其重要的传播载体之一。探究生菜叶中特异性黏附hNoV的介质，及病毒与介质结合的关键位点是揭示hNoV与生菜叶互作机制的突破口。本项目以前期利用细菌细胞表面展示体系成功构建约百倍放大的升级版假hNoV为媒介，靶向性富集生菜叶中可与不同基因型病毒衣壳蛋白特异性结合的介质（主要是糖类和蛋白质），并进行分离和鉴定，以酶解和竞争ELISA方法明确其与病毒结合的主要官能团；以定点突变试剂盒构建病毒主要衣壳蛋白受体结合域（P区）单/多点突变体库，并对P区突变体与捕获的介质进行吸附能力评估，获得病毒衣壳蛋白与生菜叶介质互作的关键氨基酸位点，定位病毒黏附生菜叶所依赖的衣壳蛋白结构域；以SPR进行吸附动力学验证。最终明确生菜叶特异性吸附hNoV的物质基础，揭示病毒与生菜叶的互作机制，为构建果蔬中该病毒污染的防控体系提供理论支撑。

人源诺如病毒（human norovirus，hNoV）是重要的食源性病毒之一。生菜是其重要传播载体之一。但是，hNoV与生菜叶的互作机制不清楚。针对上述科学问题，本项目完成了四个方面的工作内容，相关研究结果如下：（1）明确了生菜中黏附hNoV的主要介质是碳水化合物（糖类物质），蛋白质类介质的作用较小；中性（pH 6.0-8.0）低盐溶液有助于生菜叶黏附hNoV，且游离血组织抗原（hNoV的公认受体，Histo-blood Group Antigens，HBGAs）具有一定的阻断病毒黏附的效果。（2）以细菌细胞表面展示体系成功构建的升级版GI.1/GII.4型假hNoV为媒介，靶向性富集生菜叶中可与不同基因型病毒衣壳蛋白特异性结合的介质，分离并鉴定到一种由A、H和Lewis a型三种HBGAs组成的嵌合体。酶联免疫吸附试验结果显示：不同基因型hNoV与生菜中HBGAs嵌合体结合的糖类物质是相同的，但是两者互作的主要官能团存在差异，GI.1型hNoV主要结合A型HBGA，而GII.4型hNoV主要结合H型HBGA。（3）以定点突变试剂盒构建了hNoV主要衣壳蛋白受体结合域（P区）的单/多点突变体库，并对P区突变体与HBGAs嵌合体进行吸附能力评估，明确了病毒衣壳蛋白与生菜叶介质互作的关键氨基酸位点位于病毒衣壳蛋白口袋结构；P蛋白325位氨基酸是关键位点。（4）从生菜叶面分离到18株可不同程度上分泌类HBGAs物质的细菌且可以有效的结合hNoV；其中一株魔氏假单胞菌可分泌5种类HBGAs物质。魔氏假单胞菌基因组测序结果显示：该菌基因组编码了两个类岩藻糖基转移酶（fut 2和fut 3），阐明了该类细菌分泌类HBGAs的物质基础。另外，我们推测生菜表面的常在细菌可帮助hNoV黏附在生菜叶面并进行传播。上述研究结论明确了生菜叶中吸附hNoV的物质基础是类HBGAs物质，揭示病毒与生菜叶的互作机制，为构建果蔬中hNoV污染的防控体系提供理论支撑。.经过上述研究，本项目共资助发表研究性论文11篇，其中，SCI论文10篇（JAFC封面文章1篇），中文核心期刊1篇。授权国家发明专利1件。培养硕士研究生3名，博士研究生1名。