H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects1,2. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-α2,6-galactose (SAα2,6Gal), whereas the latter prefer those ending in SAα2,3Gal (refs 3–6). A conversion from SAα2,3Gal to SAα2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Webster, R. G., Peiris, M., Chen, H. & Guan, Y. H5N1 outbreaks and enzootic influenza. Emerg. Infect. Dis. 12, 3–8 (2006)
Enserink, M. Avian influenza. H5N1 moves into Africa, European Union, deepening global crisis. Science 311, 932 (2006)
Matrosovich, M. et al. Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J. Virol. 74, 8502–8512 (2000)
Rogers, G. N. & Paulson, J. C. Receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the H3 hemagglutinin based on species of origin. Virology 127, 361–373 (1983)
Rogers, G. N., Pritchett, T. J., Lane, J. L. & Paulson, J. C. Differential sensitivity of human, avian, and equine influenza A viruses to a glycoprotein inhibitor of infection: selection of receptor specific variants. Virology 131, 394–408 (1983)
Zambon, M. C. The pathogenesis of influenza in humans. Rev. Med. Virol. 11, 227–241 (2001)
Le, Q. M. et al. Avian flu: isolation of drug-resistant H5N1 virus. Nature 437, 1108 (2005)
Neumann, G. et al. Generation of influenza A viruses entirely from cloned cDNAs. Proc. Natl Acad. Sci. USA 96, 9345–9350 (1999)
Ha, Y., Stevens, D. J., Skehel, J. J. & Wiley, D. C. X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proc. Natl Acad. Sci. USA 98, 11181–11186 (2001)
Stevens, J. et al. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312, 404–410 (2006)
Eisen, M. B., Sabesan, S., Skehel, J. J. & Wiley, D. C. Binding of the influenza A virus to cell-surface receptors: structures of five hemagglutinin-sialyloligosaccharide complexes determined by X-ray crystallography. Virology 232, 19–31 (1997)
Hardy, C. T. et al. Egg fluids and cells of the chorioallantoic membrane of embryonated chicken eggs can select different variants of influenza A (H3N2) viruses. Virology 211, 302–306 (1995)
Gubareva, L. V. et al. Codominant mixtures of viruses in reference strains of influenza virus due to host cell variation. Virology 199, 89–97 (1994)
Jones, T. A., Zhou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)
Shinya, K. et al. Avian flu: influenza virus receptors in the human airway. Nature 440, 435–436 (2006)
Kuiken, T. et al. Host species barriers to influenza virus infections. Science 312, 394–397 (2006)
Connor, R. J., Kawaoka, Y., Webster, R. G. & Paulson, J. C. Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology 205, 17–23 (1994)
Clements, M. L. et al. Use of single-gene reassortant viruses to study the role of avian influenza A virus genes in attenuation of wild-type human influenza A virus for squirrel monkeys and adult human volunteers. J. Clin. Microbiol. 30, 655–662 (1992)
Subbarao, E. K., London, W. & Murphy, B. R. A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. J. Virol. 67, 1761–1764 (1993)
Shinya, K. et al. PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5N1 influenza A viruses in mice. Virology 320, 258–266 (2004)
Shinya, K. et al. Characterization of a human H5N1 influenza A virus isolated in 2003. J. Virol. 79, 9926–9932 (2005)
Totani, K. et al. Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(l-glutamic acid) backbone for inhibition of infection by influenza viruses. Glycobiology 13, 315–326 (2003)
Ha, Y., Stevens, D. J., Skehel, J. J. & Wiley, D. C. H5 avian and H9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes. EMBO J. 21, 865–875 (2002)
Otwinowski, Z. & Minor, W. in Data Collection and Processing (eds Sawyer, L., Isaacs, N. & Bailey, S.) 556–562 (SERC Daresbury Laboratory, Warrington, UK, 1993)
CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)
Brunger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)
We thank K. Wells for technical assistance, and J. Gilbert for editing the manuscript. The NIMR contributors were responsible for the structural studies and for HA sequencing. This work was supported by CREST (Japan Science and Technology Agency); by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan; by the Ministry of Health, Labour and Welfare, Japan; and by grants from the NIH, NIAID. Structural studies were supported by the UK MRC and by an International Partnership Research Award in Veterinary Epidemiology of the Wellcome Trust. Author Contributions S.Y., Y.S., T.S., M.Q.L., C.A.N., Y.S.T., Y.M., T.H., T.S., M.K, T.U., T.M., Y.L., A.H. and Y.K. were responsible for the virological studies. L.F.H., D.J.S., R.J.R., S.J.G. and J.J.S. were responsible for the structural studies.
This file contains Supplementary Figures 1–4. Supplementary Figure 1: Phylogenetic relationships of HA genes from H5N1 influenza viruses. Supplementary Figure 2: Receptor specificity of H5N1 viruses. Supplementary Figure 3: Specificity of the receptor assay. Supplementary Figure 4: Effect of HA mutations on SAα2,6Gal recognition. (PPT 6961 kb)
Supplementary Table 1: H5N1 viruses used in this study. Supplementary Table 2: Data collection and refinement statistics (DOC 82 kb)
About this article
Insights into structural and inhibitory mechanisms of low pH-induced conformational change of influenza HA2 protein: a computational approach
Journal of Molecular Modeling (2019)
Unlocking pandemic potential: prevalence and spatial patterns of key substitutions in avian influenza H5N1 in Egyptian isolates
BMC Infectious Diseases (2018)
Nature Reviews Disease Primers (2018)
Genetic and biological characterization of three poultry-origin H5N6 avian influenza viruses with all internal genes from genotype S H9N2 viruses
Archives of Virology (2018)
Mutations Driving Airborne Transmission of A/H5N1 Virus in Mammals Cause Substantial Attenuation in Chickens only when combined
Scientific Reports (2017)