Li Qianqian, Wang Dapeng, Yang David, Shan Lei, Tian Peng
Department of Bioengineering, Shanghai Institute of TechnologyShanghai, China.
Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, AlbanyCA, United States.
Front Microbiol. 2017 Sep 21;8:1746. doi: 10.3389/fmicb.2017.01746. eCollection 2017.
Histo-blood group antigens (HBGAs) are considered as receptors/co-receptors for human norovirus (HuNoV). It has been reported that binding of HuNoV-derived virus-like particles (VLPs) to HBGA-like molecules-expressing bacteria increased the stability of VLPs to heat-denaturation (HD). In this study, we tested for HBGA-like-binding-conveyed protection against HD on viral replication using Tulane virus (TV) and O86:H2 (O86:H2), with K-12 (K-12) used as a control. Expression of HBGA type B was confirmed by ELISA in O86:H2 but not in K-12. Binding of TV was confirmed by ELISA in O86:H2 (P/N = 2.23) but not in K-12 (P/N = 1.90). Pre-incubation of TV with free HBGA could completely inhibit its ability to bind to O86:H2 ( = 0.004), while producing no significant change in its ability to bind K-12 ( = 0.635). We utilized a bacterial-capture-RT-qPCR procedure to confirm that both bacterial strains were capable of binding TV, and that O86:H2 exhibited fivefold greater binding capacity than K-12. Pre-incubation of TV with free HBGA would partially inhibit the binding of TV to O86:H2 ( = 0.047). In contrast, not only did pre-incubation of TV with free HBGA not inhibit the binding of TV to K-12, binding was slightly enhanced ( = 0.13). The viral infectivity assay allowed us to conduct a direct evaluation of the ability of HBGA-like-bound bacteria to confer HD protection to TV. Prior to inoculate to LLC-MK2 cells, TV was incubated with each bacterial strain at ratios of 1:0, 1:1 and 100:1, then both partially and fully HD. The viral amplification was quantitated by RT-qPCR 48 h later. The binding of bacteria to TV reduced viral replication in a dose-dependent matter. We found that neither bound O86:H2 nor K-12 conferred protection of TV against partial or full HD conditions. Partial HD reduction of viral replication was not significantly impacted by the binding of either bacterial strain, with infectivity losses of 99.03, 99.42, 96.32, 96.10, and 98.88% for TV w/o bacteria, TV w/O86:H2 (1:1), TV w/O86:H2 (100:1), TV w/K-12 (1:1), and TV w/K-12 (100:1), respectively. Full HD reduction of viral replication was not impacted by the binding of either bacterial strain, as full loss of infectivity was observed in all cases.
组织血型抗原（HBGAs）被认为是人类诺如病毒（HuNoV）的受体/共受体。据报道，HuNoV衍生的病毒样颗粒（VLPs）与表达HBGA样分子的细菌结合可提高VLPs对热变性（HD）的稳定性。在本研究中，我们使用杜兰病毒（TV）和O86:H2菌株，以K-12菌株作为对照，测试了HBGA样结合对病毒复制过程中HD的保护作用。通过酶联免疫吸附测定（ELISA）证实O86:H2菌株表达B型HBGA，而K-12菌株不表达。通过ELISA证实TV与O86:H2菌株结合（P/N = 2.23），但不与K-12菌株结合（P/N = 1.90）。TV与游离HBGA预孵育可完全抑制其与O86:H2菌株结合的能力（P = 0.004），而其与K-12菌株结合的能力无显著变化（P = 0.635）。我们利用细菌捕获逆转录定量聚合酶链反应（bacterial-capture-RT-qPCR）方法证实两种细菌菌株均能结合TV，且O86:H2菌株的结合能力比K-12菌株高五倍。TV与游离HBGA预孵育会部分抑制TV与O86:H2菌株的结合（P = 0.047）。相反，TV与游离HBGA预孵育不仅不会抑制TV与K-12菌株的结合，反而会使结合略有增强（P = 0.13）。病毒感染性测定使我们能够直接评估HBGA样结合细菌赋予TV抗HD保护的能力。在接种到LLC-MK2细胞之前，将TV与每种细菌菌株按1:0、1:1和100:1的比例孵育，然后进行部分和完全HD处理。48小时后通过RT-qPCR对病毒扩增进行定量。细菌与TV的结合以剂量依赖方式降低病毒复制。我们发现，结合的O86:H2菌株和K-12菌株均未赋予TV对部分或完全HD条件的保护。两种细菌菌株的结合对病毒复制的部分HD降低没有显著影响，对于未与细菌孵育的TV、与O86:H2菌株（1:1）孵育的TV、与O86:H2菌株（100:1）孵育的TV、与K-12菌株（1:1）孵育的TV和与K-12菌株（100:1）孵育的TV，其感染性损失分别为99.03%、99.42%、96.32%、96.10%和98.88%。两种细菌菌株的结合对病毒复制的完全HD降低没有影响，因为在所有情况下均观察到感染性完全丧失。
