Cheng Chao, Sun Minjia, Li Jingjing, Xue Yitong, Cai Xia, Liu Jing, Wang Xiaolian, Xu Shouhong, Xie Youhua, Zhang Junqi
MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, Shanghai Institute of Infectious Disease and Biosecurity, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, People's Republic of China.
Key Laboratory for Advanced Materials and Department of Chemistry, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
Int J Nanomedicine. 2025 Feb 11;20:1789-1805. doi: 10.2147/IJN.S495399. eCollection 2025.
Human noroviruses (HuNoVs) are the main cause of non-bacterial acute gastroenteritis. Due to antigenic diversity, the discovery of ligands that can sensitively and specifically detect HuNoVs remains challenging. Limited by laboratory culture, no vaccines or drugs have been developed against HuNoVs. Here, we screened nucleic acid aptamers against the widespread HuNoV GII.4 and emerging HuNoV GII.17.
After ten rounds of sieving for HuNoV GII.4 and GII.17 virus-like particles (VLPs), eight ssDNA aptamers were generated and characterized for each genotype.
Four of the eight aptamers generated for GII.4 VLP had dissociation constants (K) less than 100 nM, and all aptamers for GII.17 VLP had K less than 10 nM. All aptamers bound to their targets in VLP concentration-dependent manner. Two aptamers (AP4-2 and AP17-4) were selected for enzyme-linked aptamer sorbent assay (ELASA) and further analysis. Binding affinity was enhanced as the concentration of both aptamer and VLPs increased. The specificity of the aptamers was verified by ELASA and dot blotting. AP4-2 and AP17-4 were able to differentiate HuNoV from other diarrhea-causing pathogens or unrelated proteins ( < 0.0001). VLP/porcine gastric mucin (PGM) binding blockade assays revealed that AP4-2 and AP17-4 blocked the binding of HuNoV VLPs to PGM. VLP internalization inhibition assays showed that at a concentration of 0.5 µM, both AP4-2 and AP17-4 effectively inhibited attachment and internalization of HuNoV VLPs into 293T cell ( < 0.05). Cell viability assays confirmed that aptamers did not induce cellular toxicity.
AP4-2 and AP17-4 showed strong affinity and specificity for their target VLPs and represent promising candidates for HuNoV capture and detection. This is the first study to demonstrate that aptamers can effectively inhibit HuNoV VLPs from binding to or entering cells, thus providing a new concept for the treatment of HuNoVs.
人诺如病毒（HuNoVs）是非细菌性急性肠胃炎的主要病因。由于抗原多样性，发现能够灵敏且特异性检测HuNoVs的配体仍然具有挑战性。受实验室培养条件限制，目前尚未开发出针对HuNoVs的疫苗或药物。在此，我们筛选了针对广泛流行的HuNoV GII.4和新出现的HuNoV GII.17的核酸适配体。
对HuNoV GII.4和GII.17病毒样颗粒（VLPs）进行十轮筛选后，针对每个基因型生成并表征了8个单链DNA适配体。
针对GII.4 VLP生成的8个适配体中有4个的解离常数（K）小于100 nM，针对GII.17 VLP的所有适配体的K均小于10 nM。所有适配体均以VLP浓度依赖性方式与其靶标结合。选择了两个适配体（AP4 - 2和AP17 - 4）用于酶联适配体吸附测定（ELASA）和进一步分析。随着适配体和VLP浓度的增加，结合亲和力增强。通过ELASA和斑点印迹验证了适配体的特异性。AP4 - 2和AP17 - 4能够区分HuNoV与其他引起腹泻的病原体或无关蛋白质（<0.0001）。VLP/猪胃粘蛋白（PGM）结合阻断试验表明，AP4 - 2和AP17 - 4阻断了HuNoV VLPs与PGM的结合。VLP内化抑制试验表明，在浓度为0.5 μM时，AP4 - 2和AP17 - 4均有效抑制HuNoV VLPs附着并内化到293T细胞中（<0.05）。细胞活力测定证实适配体不会诱导细胞毒性。
AP4 - 2和AP17 - 4对其靶标VLP表现出强亲和力和特异性，是HuNoV捕获和检测的有前景的候选物。这是第一项证明适配体可有效抑制HuNoV VLPs与细胞结合或进入细胞的研究，从而为HuNoVs的治疗提供了新的概念。
