Differences in HA receptor binding and NA substrate specificities of high and low pathogenic avian influenza viruses

Background The viral glycoproteins, HA and NA, expressed on the surface of influenza virions play an important role in determining pathogenic properties of this virus. Influenza virus infection is initiated by specific interactions between the viral HA and terminal sialic acid-containing molecules of cell surface receptors mediating the virus absorption on the target cells. The NA cleaves off the terminal sialic acid residues from the host cell promoting the release of virus progeny and preventing the formation of virion aggregates at the budding site. The functional balance between HA and NA is prerequisite of successful influenza virus replication. Human infections caused by highly pathogenic avian influenza viruses emphasize a need for the assessment of factors that allow adaptation of avian viruses (AIV) to humans. Here, in order to clear a possible role of HA and NA as well as their balanced action in pathogenic properties of AIV, we evaluated HA receptor binding and NA substrate specificities of high and low pathogenic avian influenza (HPAI and LPAI) viruses of H5N1, H5N9, H5N2, H7N7 and H9N2 subtypes for a set of synthetic sialooligosaccharides which are analogues of the natural influenza virus receptors. Methods Receptor binding specificity of HA was investigated in a direct binding assay as described previously (Matrosovich et al., 2000). In brief, 96-well plates were coated with purified virus for 16 h at 4°C. After that biotinylated sialyloligosaccharides in TN buffer containing neuraminidase inhibitor were added. Following the addition of Strept-POD and ABTS substrate solution, optical density at 405 nm was determined. The affinity constants (Kaff) were determined as sialic acid concentration (μM-1) at the point Amax/2 on Scatchard plots. The fluorescent assay for studying the substrate specificity of neuraminidase was described earlier (Mochalova et al., 2005). The method based on the a quantitative separation of neutral fluorescent-labeled product from negatively charged fluorescent-labeled uncleaved substrate using anion exchanger microcartridges. Fluorescence was measured at 485/535 nm. Virus NA specificity for each sialoside was calculated as the slope of the starting linear region of the Vo versus So curve (Vo - initial rate of the desialylation, So - initial substrate concentration). Results HA receptor binding and NA substrate oligosaccharide specificity are usually defined as ability of the viral HA or NA to distinguish the type of bond between terminal neuraminic acid and galactose residues. HPAI viruses displayed a higher (2-12 times) HA affinity and higher (2-8 times) NA hydrolytic efficiency than LPAI viruses towards trisaccharides Neu5Acα2-3Galß1-4GlcNAcß (3’SLN) and Neu5Acα2-3Galß1-3GlcNAcß (SiaLec). Furthermore, HAs of HPAI and LPAI viruses differed in their ability to discriminate non-fucosylated and fucosylated glycans, namely, 3’SLN vs. Neu5Acα2-3Galß1-4(Fucα1-3)GlcNAcß?(SiaLex). When receptor binding specificity was plotted as 3’SLN/SiaLex ratio, the values for all HPAI viruses ranged between 3 - 5 whereas for LPAI viruses these values were only about 2 or lower. We hypothesize that the optimal ratio of affinities towards 3’SLN and SiaLex is an important modulator of pathogenicity. Conclusion In summary, this study presents combined data on HA and NA specificity of HPAI and LPAI viruses. A combination of both methods allows for a rapid monitoring of changes in the OS specificity of both HA and NA. Correlation of these data with mutational analyses of the glycoprotein genes promise to be a powerful tool for the prediction of new pandemic strains.

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