Abstract
ABSTRACT Switching of receptor binding preference has been widely considered as one of the necessary mutations for avian influenza viruses, enabling efficient transmissions between human hosts. By stably overexpressing B4GalNT2 gene in MDCK cells, surface α2,3-siallylactose receptors were modified without affecting α2,6-receptor expression. The cell line MDCK-B4GalNT2 was used as a tool to screen for α2,3-receptor requirements in a panel of influenza viruses with previously characterized glycan array data. Infection of viruses with α2,3-receptor binding capability was inhibited in MDCK-B4GalNT2 cells, with the exception of A/WSN/33 (WSN). Infection with the 2009 pandemic H1N1 strains, A/California/04/2009 (Cal04) and A/Hong Kong/415742/2009 (HK09), despite showing α2,6-receptor binding, was also found to be inhibited. Further investigation showed that viral inhibition was due to a reduction in viral entry rate and viral attachment. Recombinant WSN virus with the neuraminidase (NA) gene swapped to A/Puerto Rico/8/1934 (PR8) and Cal04 resulted in a significant viral inhibition in MDCK-B4GalNT2 cells. With oseltamivir, the NA active site was found to be important for the replication results of WSN, but not Cal04.
Highlights
A switch from α2,3-sialic acid receptor binding to α2,6sialic acid receptor binding is one of the necessary steps for avian influenza viral adaptation to humans [1,2]
To ensure B4GalNT2 overexpression was functioning to convert α2,3-sailic acid receptors into Sda-like epitopes, cells were stained with MAA1, MAA2, SNA, DBA and CT-1 antibody (Sda epitopes) by ICC (Figure 2)
enzyme-linked lectin assay (ELLA) was performed to numerically measure lectin binding levels. 2 more clones of B4GalNT2 transduced MDCK (MDCK-B4GalNT2 #2 and #3) and a polyclonal YFP transduced MDCK cell line (MDCK-YFP) were used to ensure the changes in surface glycan expressions were the specific effect of transduced B4GalNT2 gene
Summary
A switch from α2,3-sialic acid receptor binding to α2,6sialic acid receptor binding is one of the necessary steps for avian influenza viral adaptation to humans [1,2]. This is in concert with the sialic acid receptor distribution in the upper respiratory tracts of birds and humans [3,4,5]. Epithelial cells in the human and ferret upper respiratory tract both bound to the lectins Maackia amurensis agglutinin I (MAA1, α2,3–N glycans) and Sambuccus nigra agglutinin (SNA, α2,6–glycans), with additional binding in the human respiratory tract to Maackia amurensis agglutinin II (MAA2, α2,3–O glycans), epithelial cells in ferret trachea and lung were found to possess abundant Sda epitopes
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