Nucleic Acid Aptamers for Human Norovirus GII.4 and GII.17 Virus-Like Particles (VLPs) Exhibit Specific Binding and Inhibit VLPs from Entering Cells.

PURPOSE

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.

METHODS

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.

RESULTS

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.

CONCLUSION

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.