果蔬表面HBGAs表达细菌对诺如病毒存活及持续性感染的影响

Fresh produces, such as lettuce and strawberry, are high risk foods for human norovirus (NoV) contamination. To help control this pathogen in fresh produces, a better understanding of the dynamics of HuNoV binding to fresh produces needs to be established. Since HuNoV have been shown to bind in a facilitated manner such as in association with microflora, we examined the effect of fresh produces commensal bacteria on the NoV biological characteristics and its role in the attachment of NoV to fresh produces by using virus-like particles (VLPs) of a human NoV strains. In this study, the effects of commensal bacteria on the NoV biological characteristics were evaluated under different conditions during the harvest. And then the native bacteria contained HBGA-like substances were isolated by using a biopanning technique with anti-HBGA antibodies. After that the NoVLPs bound to the extracellular polymeric substances (EPS) of commensal bacteria, where the HBGA-like substances were localized, were analyzed by Immuno-TEM method. And the location of NoVLPs-Bacteria in lettuce and strawberry was visualized using a fluorescence-based QuantumDots (Q-Dots) assay and confocal microscopy. To elucidate the specific mechanisms governing the binding of HuNoV to the commensal bacteria surface structures, subsequently, the roles of HBGA-like substances in the native bacteria EPS binding to NoVLPs were analyzed by enzyme-linked immunosorbent assay (ELISA). This study will support the hypothesis that lettuce and strawberry contain HBGA-like substances bacteria that mediate specific HuNoV binding interactions. This increase in our understanding of how and why HuNoVs attach to lettuce and strawberry surfaces can aid in the development of effective removal strategies such as targeted washes, as well as provide a strong basis for resolving the binding dynamics of HuNoVs to other commonly contaminated foods.

诺如病毒(Norovirus,NoV)是通过食源性感染的最大病毒因子，常吸附于食物表面传播。细菌可影响NoV吸附能力。生鲜果蔬常涉及NoV暴发，深入研究果蔬表面细菌对NoV生物特性影响将为控制果蔬NoV污染提供有效方法和思路。本项目针对常涉及NoV暴发的生菜和草莓，研究其共生细菌对NoV吸附能力、存活和持续性感染等生物特性的影响及其机理。具体包括模拟采收前后不同生长环境或处理条件下果蔬菌群对NoV生物特性的影响；利用NoV能够吸附于一些表达组织血型抗原(Histo-Blood Group Antigens,HBGAs)细菌的特性，构建菌群中HBGAs表达细菌筛选方法；利用免疫透射电镜技术观察细菌表面NoV病毒样颗粒(Virus-Like Particles,VLPs)吸附位点；HBGAs表达细菌和VLPs共同存在下，利用EZ-Link生物素标记法分析VLPs在果蔬表面的吸附位点，并提取细菌中NoV结合受体，深入探索细菌影响NoV吸附作用的分子机理。本项目将为解决果蔬中NoV的污染问题提供相关科学依据。

诺如病毒(Noroviruses, NoV)是通过食源性感染的最大病毒因子，常吸附于新鲜果蔬（生菜、草莓）等食物传播。细菌可影响NoV吸附能力。本项目针对常涉及NoV暴发的生菜，研究其共生细菌对NoV吸附能力、存活和持续性感染等生物特性的影响及其机理。首先，通过高通量测序对杭州市某农田生菜和土壤中的核心菌群进行分析，并通过分离培养，筛选到25株组织血型抗原（Histo-blood group antigens, HBGAs）阳性细菌；其次，我们发现在高温、紫外照射等条件下，某些生菜HBGAs阳性细菌对NoV的吸附强度、污染时间及病毒外壳的稳定性有一定的影响；我们以其中的一株HBGAs阳性细菌SC015为例，进一步利用透射电镜观察NoV与HBGAs阳性细菌的结合位点，确定了细菌HBGAs成分可能的分布部位是细菌胞外分泌物EPS；再利用大肠杆菌表达的P蛋白颗粒捕获细菌HBGAs受体类似物，分析HBGAs受体类似物的主要单糖组成是岩藻糖、阿拉伯糖和葡萄糖，这与以报道的大多数HBGAs受体组成成分类似。最后，模拟不同高温环境下，研究了生菜细菌SC015的HBGAs受体类似物对GⅡ.4型NoVs稳定性的影响。结果发现随着温度的增加HBGAs受体类似物EPS增强了NoVs的稳定性。以上结果表面某些HBGAs阳性细菌可能影响了生菜上NoVs的食源性传播，为果蔬表面NoVs的预防与清除提供理论依据。