Modified Sialic Acids on Mucus and Erythrocytes Inhibit Influenza A Virus Hemagglutinin and Neuraminidase Functions.

Sialic acids (Sia) are the primary receptors for influenza viruses and are widely displayed on cell surfaces and in secreted mucus. Sia may be present in variant forms that include O-acetyl modifications at C-4, C-7, C-8, and C-9 positions and N-acetyl or N-glycolyl at C-5. They can also vary in their linkages, including α2-3 or α2-6 linkages. Here, we analyze the distribution of modified Sia in cells and tissues of wild-type mice or in mice lacking CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme, which synthesizes N-glycolyl (Neu5Gc) modifications. We also examined the variation of Sia forms on erythrocytes and in saliva from different animals. To determine the effect of Sia modifications on influenza A virus (IAV) infection, we tested for effects on hemagglutinin (HA) binding and neuraminidase (NA) cleavage. We confirmed that 9-O-acetyl, 7,9-O-acetyl, 4-O-acetyl, and Neu5Gc modifications are widely but variably expressed in mouse tissues, with the highest levels detected in the respiratory and gastrointestinal (GI) tracts. Secreted mucins in saliva and surface proteins of erythrocytes showed a high degree of variability in display of modified Sia between different species. IAV HAs from different virus strains showed consistently reduced binding to both Neu5Gc- and O-acetyl-modified Sia; however, while IAV NAs were inhibited by Neu5Gc and O-acetyl modifications, there was significant variability between NA types. The modifications of Sia in mucus may therefore have potent effects on the functions of IAV and may affect both pathogens and the normal flora of different mucosal sites.IMPORTANCE Sialic acids (Sia) are involved in numerous different cellular functions and are receptors for many pathogens. Sia come in chemically modified forms, but we lack a clear understanding of how they alter interactions with microbes. Here, we examine the expression of modified Sia in mouse tissues, on secreted mucus in saliva, and on erythrocytes, including those from IAV host species and animals used in IAV research. These Sia forms varied considerably among different animals, and their inhibitory effects on IAV NA and HA activities and on bacterial sialidases (neuraminidases) suggest a host-variable protective role in secreted mucus.

Influenza Viruses in Mice: Deep Sequencing Analysis of Serial Passage and Effects of Sialic Acid Structural Variation.

Influenza A viruses have regularly jumped to new host species to cause epidemics or pandemics, an evolutionary process that involves variation in the viral traits necessary to overcome host barriers and facilitate transmission. Mice are not a natural host for influenza virus but are frequently used as models in studies of pathogenesis, often after multiple passages to achieve higher viral titers that result in clinical disease such as weight loss or death. Here, we examine the processes of influenza A virus infection and evolution in mice by comparing single nucleotide variations of a human H1N1 pandemic virus, a seasonal H3N2 virus, and an H3N2 canine influenza virus during experimental passage. We also compared replication and sequence variation in wild-type mice expressing N-glycolylneuraminic acid (Neu5Gc) with those seen in mice expressing only N-acetylneuraminic acid (Neu5Ac). Viruses derived from plasmids were propagated in MDCK cells and then passaged in mice up to four times. Full-genome deep sequencing of the plasmids, cultured viruses, and viruses from mice at various passages revealed only small numbers of mutational changes. The H3N2 canine influenza virus showed increases in frequency of sporadic mutations in the PB2, PA, and NA segments. The H1N1 pandemic virus grew well in mice, and while it exhibited the maintenance of some minority mutations, there was no clear evidence for adaptive evolution. The H3N2 seasonal virus did not establish in the mice. Finally, there were no clear sequence differences associated with the presence or absence of Neu5Gc.IMPORTANCE Mice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts, we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and that the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not affect influenza virus evolution in this model. Overall, our results show that while mice provide a useful animal model for influenza virus pathology, host passage evolution will vary depending on the specific virus tested.

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

Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C-7, C-8, and C-9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. Since Sia or their variant forms are receptors for influenza A, B, C, and D viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. Although they were expressed on the cell surfaces of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1 to 2% of total Sia in these cell lines. We knocked out and overexpressed the sialate O-acetyltransferase gene (CasD1) and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, overexpression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C and D infection but had no obvious effect on influenza A and B infection.IMPORTANCE Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here, we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.