Glycoconjugate Journal

, Volume 27, Issue 3, pp 321–327 | Cite as

Shift in oligosaccharide specificities of hemagglutinin and neuraminidase of influenza B viruses resistant to neuraminidase inhibitors

  • Larisa Mochalova
  • Rick Bright
  • Xiyan Xu
  • Elena Korchagina
  • Alexander Chinarev
  • Niсolai Bovin
  • Alexander Klimov


Influenza virus neuraminidase inhibitors (NAIs), currently used as anti-influenza drugs, can lead to the appearance of drug-resistant variants. Resistance to NAIs appears due to mutations in the active site of the neuraminidase (NA) molecule that decrease the NA enzymatic activity and sometimes in the hemagglutinin (HA) that decrease its affinity for cell receptors and, therefore, reduce the requirement for NA activity in releasing mature virions from infected cells. Using a set of sialo-oligosaccharides, we evaluated changes in the receptor-binding specificity of the HA and substrate specificity of the NA of influenza B viruses that had acquired resistance to NAIs. The oligosaccharide specificity of two pairs of field influenza B viruses, namely: i) B/Memphis/20/96 and its NAI-resistant variant, B/Memphis/20-152K/96, containing mutation R152K in the NA and 5 amino acid substitutions in the HA1, and ii) B/Hong Kong/45/2005 and its NAI-resistant variant B/Hong Kong/36/2005, containing a single R371K mutation in the NA, was evaluated. Wild type viruses bound strictly to a “human type” receptor, α2-6-sialo-oligosaccharide 6`SLN, but desialylated it is approximately 8 times less efficiently than the α2-3 sialosaccharides. Both drug-resistant viruses demonstrated the ability to bind to “avian type” receptors, α2-3 sialo-oligosaccharides (such as 3`SLN), whereas their affinity for 6`SLN was noticeably reduced in comparison with corresponding wild type viruses. Thus, the development of the NAI resistance in the studied influenza B viruses was accompanied by a readjustment of HA-NA oligosaccharide specificities.


Influenza B virus Neuraminidase Neuraminidase inhibitor Drug resistance Hemagglutinin Receptor-binding specificity Neuraminidase substrate specificity 



4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid




hemagglutinating unit


inhibition of hemagglutination reaction




neuraminidase activity inhibitor


N-acetylneuraminic acid


oligosaccharide-polyacrylamide conjugate


receptor-binding site










  1. 1.
    Wagner, R., Matrosovich, M., Klenk, H.-D.: Functional balance between hemagglutinin and neuraminidase in influenza virus infections. Rev. Med. Virol. 12, 159–166 (2002)CrossRefPubMedGoogle Scholar
  2. 2.
    Matrosovich, M., Matrosovich, T., Gray, T., Roberts, N.A., Klenk, H.-D.: Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J. Virol. 78, 12665–12667 (2004)CrossRefPubMedGoogle Scholar
  3. 3.
    Kaverin, N.V., Gambaryan, A.S., Bovin, N.V., Rudneva, I.A., Shilov, A.A., Khodova, O.M., Varich, N.L., Sinitsin, B.V., Makarova, N.V., Kropotkina, E.A.: Postreassortment changes in influenza A virus hemagglutinin restoring HA-NA functional match. Virology 244, 315–321 (1998)CrossRefPubMedGoogle Scholar
  4. 4.
    Baigent, S.J., McCauley, J.W.: Glycosylation of haemagglutinin and stalk-lenth of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture. Virus Res. 79, 177–185 (2001)CrossRefPubMedGoogle Scholar
  5. 5.
    Mitnual, L.J., Matrosovich, M.N., Castrussi, M.R., Tuzikov, A.B., Bovin, N.V., Kobasa, D., Kawaoka, Y.: Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus. J. Virol. 74, 6015–6020 (2000)CrossRefGoogle Scholar
  6. 6.
    Rudneva, I.A., ll’yushina, N.A., Shilov, A.A., Varich, N.L., Sinitsyn, B.V., Kropotkina, E.A., Kaverin, N.V.: Functional interaction of the influenza virus glycoproteins. Mol. Biol. (Mosk.) 37, 31–36 (2003)CrossRefGoogle Scholar
  7. 7.
    Gimsa, U., Grötzinger, I., Gimsa, J.: Two evolutionary strategies of influenza viruses to escape host non-specific inhibitors: alteration of hemagglutinin or neuraminidase specificity. Virus Res. 42, 127–135 (1996)CrossRefPubMedGoogle Scholar
  8. 8.
    Matrosovich, M.N., Klenk, H.-D., Kawaoka, Y.: Receptor specificity, host-range, and pathogenicity of influenza viruses. In: Kawaoka, Y. (ed.) Influenza virology: Current topics. Caister Academic, Norfolk (2006)Google Scholar
  9. 9.
    Gubareva, L.V., Hyden, F.G.: M2 and neuraminidase inhibitors: anti-influenza activity, mechanisms of resistance, and clinical effectiveness. In: Kawaoka, Y. (ed.) Influenza virology: Current topics. Caister Academic, Norfolk (2006)Google Scholar
  10. 10.
    Ilyushina, N.A., Rudneva, I.A., Shilov, A.A., Klenk, H.-D., Kaverin, N.V.: Postreassortment changes in a model system: HA-NA adjustment in an H3N2 avian-human reassortant influenza virus. Arch. Virol. 150, 1327–1338 (2005). doi:10.1007/s00705-005-0490-4 CrossRefPubMedGoogle Scholar
  11. 11.
    Shtyrya, Y., Mochalova, L., Voznova, G., Rudneva, I., Shilov, A., Kaverin, N., Bovin, N.: Adjustment of receptor-binding and neuraminidase substrate specificities in avian-human reassortant influenza viruses. Glycoconj. J. 26, 99–109 (2009)CrossRefPubMedGoogle Scholar
  12. 12.
    Wagner, R., Wolf, T., Herwig, A., Pleschka, S., Klenk, H.-D.: Interdependence of hemagglutinin glycosilation and neuraminidase as regulators of influenza growth: a study by reverse genetics. J. Virol. 74, 6316–6323 (2000)CrossRefPubMedGoogle Scholar
  13. 13.
    Hughes, M., Matrosovich, M., Rodgers, M., McGregor, M., Kawaoka, Y.: Influenza A viruses lacking sialidase activity can undergo multiple cycles of replication in cell culture, eggs, or mice. J. Virol. 74, 5206–5212 (2000)CrossRefPubMedGoogle Scholar
  14. 14.
    Gulati, S., Smith, D.F., Air, J.M.: Deletions of neuraminidase and resistance to oseltamivir may be a consequence of restricted receptor specificity in recent H3N2 influenza viruses. Virology J. 6, 22 (2009). doi:10.1186/1743-422X-6-22 CrossRefGoogle Scholar
  15. 15.
    Sheu, T.G., Deyde, V.M., Okomo-Adhiambo, M., Garten, R.J., Xu, X., Bright, R.A., Butler, E.N., Wallis, T.R., Klimov, A.I., Gubareva, L.V.: Surveillance for neuraminidase inhibitor resistance among human influenza A and B viruses circulating worldwide from 2004 to 2008. Antimicrob. Agents Chemother. 52, 3284–3292 (2008). doi:10.1128/AAC.00555-08 CrossRefPubMedGoogle Scholar
  16. 16.
    Dharan, N.L., Gubareva, L.V., Meyer, J.J., Okomo-Adhiambo, M., et al.: Infections with oseltamivir-resistant influenza A(H1N1) virus in the United States. JAMA (2009). doi:10.1001/jama.2009.294 PubMedGoogle Scholar
  17. 17.
    Gubareva, L.V., Matrosovich, M.N., Brenner, M.K., Bethell, R.C., Webster, R.G.: Evidence for zanamivir resistance in an immunocompromised child infected with influenza B virus. J. Infect. Dis. 178, 1257–1262 (1998)CrossRefPubMedGoogle Scholar
  18. 18.
    Mochalova, L., Gambaryan, A., Romanova, J., Tuzikov, A., Chinarev, A., Katinger, D., Katinger, H., Egorov, A., Bovin, N.: Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs. Virology 313, 473–480 (2003)CrossRefPubMedGoogle Scholar
  19. 19.
    Mochalova, L., Kurova, V., Styrya, Y., Korchagina, E., Gambaryan, A., Belyanchikov, I., Bovin, N.: Oligosaccharide specificity of influenza H1N1 virus neuraminidases. Arch. Virol. 152, 2047–2057 (2007)CrossRefPubMedGoogle Scholar
  20. 20.
    Mochalova, L.V., Korchagina, E.Y., Kurova, V.S., Styria, J.A., Gambaryan, A.S., Bovin, N.V.: Fluorescent assay for studying the substrate specificity of neuraminidase. Analyt. Biochem. 34, 190–193 (2005)CrossRefGoogle Scholar
  21. 21.
    Janakiraman, M.N., White, C.L., Laver, W.G., Air, G.M., Luo, M.: Structure of influenza virus neuraminidase B/Lee/40 complexed with sialic acid and dehydro analog at 1.8 Å resolution: implications for the catalytic mechanism. Biochem. 33, 8172–8179 (1994)CrossRefGoogle Scholar
  22. 22.
    Matrosovich, M.N., Mochalova, L.V., Marinina, V.P., Byramova, N.E., Bovin, N.V.: Synthetic polymeric sialoside inhibitors of influenza virus receptor-binding activity. FEBS Lett. 272, 209–212 (1990)CrossRefPubMedGoogle Scholar
  23. 23.
    Gambarayn, A.S., Marinina, V.P., Tuzikov, A.B., Bovin, N.V., Rudneva, I.A., Sinitsin, B.V., Shilov, A.A., Matrosovich, M.N.: Effect of host-dependent glycosylation of hemagglutinin on receptor-binding properties of H1N1 human influenza A virus grown in MDCK cells and in embryonated eggs. Virology 247, 170–177 (1998)CrossRefGoogle Scholar
  24. 24.
    Gambaryan, A., Robertson, J.S., Matrosovich, M.N.: Effect of egg-adaptation on the receptor-binding properties of human influenza A and B viruses. Virology 258, 232–239 (1999)CrossRefPubMedGoogle Scholar
  25. 25.
    Xu, G., Horiike, G., Suzuki, T., Miyamoto, D., Kumihashi, H., Suzuki, Y.: A novel strain, B/Gifu/2/73, differs from other influenza B viruses in the receptor binding specificities toward sialo-sugar chain linkage. Biochem. Biophys. Res. Communs 224, 815–818 (1996)CrossRefGoogle Scholar
  26. 26.
    Wang, Q., Tian, X., Chen, X., Ma, J.: Structural basis for receptor specificity of influenza B virus hemagglutinin. Proc. Nat. Acad. Sci. 104, 16874–16879 (2007)CrossRefPubMedGoogle Scholar
  27. 27.
    Ohuchi, M., Ohuchi, R., Feldmann, A., Klenk, H.-D.: Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety. J. Virol. 71, 8377–8384 (1997)PubMedGoogle Scholar
  28. 28.
    Mishin, V.P., Novikov, D., Hayden, F.G., Gubareva, L.V.: Effect of hemagglutinin glycosylation on influenza virus susceptibility to neuraminidase inhibitor. J. Virol. 79, 12416–12424 (2005)CrossRefPubMedGoogle Scholar
  29. 29.
    Luo, C., Nobusawa, E., Nakajima, K.: An analysis of the role of neuraminidase in the receptor-binding activity of influenza B virus: the inhibitory effect of zanamivir on haemadsorption. J. Gen. Virol. 80, 2969–2976 (1999)PubMedGoogle Scholar
  30. 30.
    Gubareva, L.V., Nedyalkova, M.S., Novikov, D.V., Murti, K.G., Hoffmann, E., Hayden, F.G.: A release-competent influenza A virus mutant lacking the coding capacity for the neuraminidase active site. J. Gen. Virol. 83, 2683–2692 (2002)PubMedGoogle Scholar
  31. 31.
    Gambaryan, A.S., Tuzikov, A.B., Piskarev, V.E., Yamnikova, S.S., Lvov, D.K., Robertson, J.S., Bovin, N.V., Matrosovich, M.N.: Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6`-sialyl(N-acetyllactosamine). Virology 232, 345–350 (1997)CrossRefPubMedGoogle Scholar
  32. 32.
    Romanova, J., Katinger, D., Ferko, B., Voglauer, R., Mochalova, L., Bovin, N., Lim, W., Katinger, H., Egorov, A.: Distinct host range of influenza H3N2 virus isolates in Vero and MDCK cells is determined by cell specific glycosylation pattern. Virology 307, 90–97 (2003)CrossRefPubMedGoogle Scholar
  33. 33.
    Kumari, K., Gulati, S., Smith, D.F., Gulati, U., Cummings, R.D., Air, G.M.: Receptor-binding specificity of recent human H3N2 influenza viruses. Virology J. 4, 42 (2007). doi:10.1186/1743-422X-4-42 CrossRefGoogle Scholar
  34. 34.
    Kobasa, D., Kodihalli, S., Luo, M., Castrucci, M.R., Donatelli, I., Suzuki, Y., Suzuki, T., Kawaoka, Y.: Amino acid resides contributing to the substrate specificity of the influenza A virus neuraminidase. J. Virol. 73, 6743–6751 (1999)PubMedGoogle Scholar
  35. 35.
    Shtyrya, Y., Mochalova, L., Gambarayn, A., Korchagina, E., Xu, X., Klimov, A., Bovin, N.: Neuraminidases of H9N2 influenza viruses isolated from different hosts display various substrate specificity. In: Options for the control of influenza VI, pp. 64–65. International Medical, Atlanta (2008). Toronto, Ontario, Canada, 17–23 June 2007Google Scholar
  36. 36.
    Barros Jr., J.F., Alviano, D.S., Silva, M.H., Wigg, M.D., Alviano, C.S., Schauer, R., Couceiro, J.N.S.S.: Characterization of sialidase from an influenza A (H3N2) virus strain: kinetic parameters and substrate specificity. Intervirology 46, 199–206 (2003). doi:10.1159/000072428 CrossRefGoogle Scholar
  37. 37.
    Gulati, U., Wu, W., Gulati, S., Kumari, K., Waner, J.L., Air, J.M.: Mismatched hemagglutinin and neuraminidase specificities in recent human H3N2 influenza viruses. Virology 339, 12–20 (2005). doi:10.1016/j.virol.2005.05.009 CrossRefPubMedGoogle Scholar
  38. 38.
    Matrosovich, M., Klenk, H.-D.: Natural and synthetic sialic acid-containing inhibitors of influenza virus receptor-binding. Rev. Med. Virol. 13, 85–97 (2003). doi:10.1002/rmv.372 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Larisa Mochalova
    • 1
    • 2
  • Rick Bright
    • 3
  • Xiyan Xu
    • 3
  • Elena Korchagina
    • 1
  • Alexander Chinarev
    • 1
  • Niсolai Bovin
    • 1
  • Alexander Klimov
    • 3
  1. 1.Russian Academy of SciencesShemyakin and Ovchinnikov Institute of Bioorganic ChemistryMoscowRussia
  2. 2.A.N. Belozersky Institute of Physico-Chemical BiologyM.V. Lomonosov Moscow State UniversityMoscowRussia
  3. 3.Virus Surveillance and Diagnosis Branch, Influenza DivisionNCIRD, CDCAtlantaUSA

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