Generation Of A MDCK Cell Line Stably Expressing Chicken β-galactoside α-2,3-sialyltransferase I (ST3GAL I)

Influenza A and B viruses bind to sialyloligosaccharides on host cell surface glycolipids or glycoproteins via the hemagglutinin(HA) protein, a surface spike protein on virions. Avian influenza viruses preferentially bind to sialyloligosaccharides containing terminal N-acetyl sialic acid linked to galactose by anα-2,3-linkage (NeuAcα-2,3-Gal), while human influenza viruses mainly bind to those containing anα-2,6-linkage (NeuAcα-2,6-Gal). Thus, viral receptor specificity and the predominant type of sialic acid-galactose linkage in sialyloligosaccharides in host epithelial cells are critical determinants of the extent of viral replication in different hosts.H5 and H9 avian ifluenza viruses are two major subtypes which impact on poultry industry heavly in China, and obtained the infectivity to mammalian host cells during evolutionary process, made huge significance in public healty. Chicken embryo culture is the most sensitive system for isolation and propagating of avian influenza viruses, while the inoculation in chicken embryo may change the property of infectivity and receptor binding to mammalian host cells. Both NeuAcα-2,3-Gal and NeuAcα-2,6-Gal are present in MDCK cells, for this reason, MDCK cell is extensively used for isolaton and propagation of influenza viruses in the laboratory, but the receptor abundance is low. So far, H5N1 and H9N2 avian influenza viruses still keep the property of preferentially binding to sialyloligosaccharides by anα-2,3-linkage (NeuAcα-2,3-Gal). The variation of the affinity to receptor and the low abundance of receptor on MDCK cells surface causes suboptimal growth in some isolates, thus, making barriers on vaccine development and anti-virus drug screen.Raise the abundance ofα-2,3-linkage receptors may solve these problems. So we generated a MDCK cell line stably expressing beta-galactoside alpha-2,3-sialyltransferase I (ST3GAL I) by transfection of MDCK cells with cDNA of chicken ST3GAL I, whose product catalyzesα-2,3-silaylation of galactose on glycoproteins, and then raises the abundance ofα-2,3-linkage receptors, named MDCK-ST3GAL I. As we expected, MDCK-ST3GAL I cells expressed higher amounts of 2,3-linked receptors than parent MDCK cells, by FACS analysis using digoxigenin(DIG)-labeled lectins Maackia amurensis agglutinin (MAA) specific for 3-linked sialic acid as an antiboty, and rhodamine labeled anti digoxigenin antibidy as a secondary antibody. Two H5N1 and five H9N2 avain influenza viruses used for replicaton test grew to higher titers in MDCK-ST3GAL I cells than in parent MDCK cells. In plaque formation assay, both H5N1 and H9N2 avain influenza viruses formed larger, clearer and more amount of plaques in MDCK-ST3GAL I cells than in parent MDCK cells.In summary, the generated MDCK-ST3GAL I cell line raises the abundance ofα-2,3-linkage receptors on MDCK cell surface by reinforcing the ability of catalyzingα-2,3-silaylation of galactose on glycoproteins via ST3GAL I. This cell line significantly improved the ability of propagation and reproduction in some H5 and H9 avian influenza virus isolates. It provids a useful tool for studying the binding affinity of avian influenza viruses toα-2,3-linkage receptors, and useful not only for monitoring the mutation of avain influenza virus's receptor binding affinity but also for selecting anti-virus drugs, neutralization assay and even for large scale production of cell culture avain influenza vaccines.