We found that the expression

of cell surface SCARB2 selleck products was slightly increased after neuraminidase treatment, and neuraminidase treatment reduced virus binding to RD and SK-N-SH cells in a dose-dependent manner. In addition, the replication of virus was decreased because the binding of EV71-GFP to RD cells was reduced after neuraminidase treatment. These results indicated that sialylation on cell surface should be involved in the attachment and infection of EV71. As long as there are two major glycosidic linkages between sialic acid with galactose, we applied the lectin competition assay to characterize the binding preference of EV71 to RD and SK-H-SN cells. Not surprisingly, the binding of EV71 was restrained by both lectins on RD and SK-H-SN cells. Both cell surface α2-3- and α2-6-linked sialosides were participated in the binding of EV71 to host cells. The replication of virus was also dropped because the interaction of EV71-GFP to RD cells was blocked by MAA or SNA. These observations Epacadostat can also be found in the infection of other Picornaviridae viruses such as human rhinovirus 87, encephalomyocarditis virus, and hepatitis A virus [28]. Then, fetuin/asialofetuin blocking assay was performed and the result indicated that sialylated glycoproteins, such as fetuin, lactoferrin and milk proteins, were inhibitors of EV71 infection [24, 25, 29]. In order to further identify the carbohydrate epitopes for EV71 infection, viral particles

and recombinant viral capsid protein were subjected to carbohydrate solution microarray analysis. But, we could not observe any positive binding signal for viral particles or recombinant VP1 protein. It might be because we don’t have sufficient sialylated epitopes in our microarray library. Further investigations are in progress (collaborate with CFG). To further characterize the role of sialylation on EV71 cellular receptor, we isolated cell membrane sialylated glycoproteins by lectin affinity chromatography. LAC was a common and useful tool for proteomic and glycomic analysis [41–45]. For about instance, Butterfield

et al. enriched and analyzed abnormal glycoproteins from brain of Alzheimer disease patient by using LAC [41]. Alvarez-Manilla and colleagues also identified potential glycobiomarkers from embryonic stem cells with LAC technology [43]. Hence, sialylated membrane proteins were purified with MAA/SNA lectin-agarose column from RD cell membrane extractions. Then, the purified glycoproteins were treated with neuraminidase to remove the effect of sialic acid. The desialylated glycoproteins were subjected to immunoprecipitation assay that pulled down proteins specifically interacted with EV71. Not surprisingly, SCARB2 was observed in western blotting of LAC purified fraction, neuraminidase treated fraction, as well as the EV71 immunoprecipitated fraction. It should be noted that the position of band in lane 4 (EV71 immunoprecipitated fraction) was inconsistent with band in lane 3.