Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses

Barnard, Karen N.; Wasik, Brian R.; LaClair, Justin R.; Buchholz, David W.; Weichert, Wendy S.; Alford-Lawrence, Brynn K.; Aguilar, Hector C.; Parrish, Colin R.

Virus infection assays. For infection in HEK-293 cells with IAV, the cells were seeded on coverslips and inoculated with a multiplicity of infection (MOI) of 0.1. Coverslips of inoculated cells were fixed at 24 h, stained for virus using an anti-NP antibody, and costained with DAPI. The percentages of infected cells were determined by imaging coverslips and counting the infected cells per field. Statistical analyses were performed in GraphPad Prism software. For infection in MDCK cells with the influenza A, B, C, and D viruses, cells were seeded into 96-well plates and inoculated for 48 h. The cells were then fixed and stained with a mouse anti-influenza A NP antibody, a mouse anti-influenza B NP antibody (Abcam), a polyclonal mouse anti-influenza C antibody (a gift from Peter Palese, Icahn School of Medicine at Mount Sinai), or a polyclonal rabbit anti-influenza D antibody (a gift from Feng Li, South Dakota State University). The cells were imaged using a Nikon TE300 fluorescence microscope. Images were analyzed for relative infected cell counts using ImageJ (NIH and LOCI, University of Wisconsin).