| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 98 === Enterovirus 71 (EV71), an unusual human pathogen of the family Picornaviridae, is the major agent cause of hand-foot-and-mouth disease (HFMD) associated with severe neurological disease. The mechanisms of virus attachment, invasion and virulence determinants of EV71 are still unclear. Recent studies showed that EV71 recognizes not only scavenger receptor B2 (SCARB2) but also human P-selectin glycoprotein ligand-1 as cellular receptors depended on difference cell types or virus strains. However, the infection of EV71 could not be inhibited by specific antibody entirely, suggesting that these might be other pathway or receptor for virus infection during early stage. Because cell surface glycans have been demonstrated to involve in the infection of viruses including influenza virus, adenovirus, and enterovirus 70, we want to investigate the role of cell surface glycans during the infection of EV71. First, we expressed α2,3- and α2,6-sialyltransferases and subjected the enzymes for producing eight sialylated glycans. Then, we analyzed the carbohydrate binding property of purified the fourth passage of mouse-adapted EV71 strain (MP4) by glycan microarray. The glycan binding results indicated that cell surface sialyl Lewis x (sLex) might play important roles on mechanisms of virus infection. In order to explore the significance of cell surface N-linked, O-linked, and sialylated glycans during EV71 binding and infection, we had treated RD cells with tunicamycin, benzyl-α-GalNAc or neuraminidase and quantified the bound of amplified virus by ELISA assay, flow cytometry, real-time PCR, and fluorescence microscopy. The results showed that the cell surface sialic acid containing biomolecules were essential for EV71 binding and infection. In addition, interactions between virus and RD cells could be inhibited by Sambucus nigra lectin (SNA, NeuAcα2-6 glycan binding lectin) and Maackia amurensis lectin (MAA, NeuAcα2-3 glycan binding lectin) in dose dependent manner. In conclusion, sialylated glycans on RD cell surface played a critical role during EV71 binding and infection.
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