1 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Saitama 332-0012, Japan
3 Laboratory of Veterinary Microbiology, Division of Veterinary Science, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan
4 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA
Correspondence
Taisuke Horimoto
horimoto{at}ims.u-tokyo.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
A figure showing the haemadsorption of the wild-type and selected mutant HAs is available as supplementary data in JGV Online.
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
The receptor-binding preference of influenza viruses correlates with the animal species from which the viruses are isolated; human isolates preferentially bind to the terminal sialic acid of glycoprotein and glycolipid receptors with 2,6 linkages to galactose (SA
2,6Gal), whereas avian isolates prefer
2,3 linkages (SA
2,3Gal) (Connor et al., 1994
; Couceiro et al., 1993
; Ito et al., 1998
; Rogers & D'Souza, 1989
). Alteration in receptor specificity may have occurred when the HA gene of an avian virus was introduced into humans, resulting in the 1957 (Asian influenza) and in the 1968 (Hong Kong influenza) pandemics, since the earliest viruses available from these pandemics recognize SA
2,6Gal (Matrosovich et al., 2000
). Interestingly, the index H5N1 human isolate (A/Hong Kong/156/97) from the 1997 outbreak preferentially recognizes the SA
2,3Gal receptor (Matrosovich et al., 1999
; Ha et al., 2001
), suggesting that the change in receptor preference may not be required for primary human infection by avian influenza viruses.
To understand further the bird-to-human transmission of influenza virus that occurred in 1997, we compared receptor specificity between the Hong Kong virus and a virulent avian H5 virus by using an assay that has not previously been used to study the 1997 human isolate.
We cloned the cDNA of the HA genes from the index human isolate in the Hong Kong 1997 outbreak, A/Hong Kong/156/97 (HK/97; H5N1) (Claas et al., 1998; Subbarao et al., 1998
), and from the virulent avian virus, A/turkey/Ontario/7732/66 (Ty/Ont; H5N9) (Horimoto & Kawaoka, 1994
), into the pCAGGS/MCS expression vector, which contains the chicken
-actin promoter (Kobasa et al., 1997
; Niwa et al., 1991
). When COS-7 cells were transfected with these plasmids (1 µg plasmid per well of a six-well plate) HAs were expressed on the cell surface as detected by immunostaining with a pool of anti-H5 monoclonal antibodies (61B2, 61E2, 81E5; the latter two react with the HA2 portion of the HA; Horimoto et al., 2004
) (see supplementary figure in JGV Online). A haemadsorption assay was performed by using human and chicken red blood cells (RBCs). We found that both human and chicken RBCs adsorbed cells expressing HK/97 HA, but only chicken RBCs adsorbed cells expressing Ty/Ont HA (Fig. 3
). These data suggest that receptor recognition differs between these two viruses. However, both RBCs bound to cells expressing HAs from other virulent avian viruses including A/turkey/Ireland/1378/85 (H5N8) HA (data not shown). We therefore focused on Ty/Ont HA to analyse further the properties of the HK/97 HA. Human and chicken RBCs contain both SA
2,3Gal and SA
2,6Gal glycoconjugates (Ito et al., 1997
; Medeiros et al., 2001
); however, our results indicate a quantitative and/or qualitative difference in the relative proportions of these cell surface molecules in the RBCs of humans compared to chickens.
|
|
|
We next tested whether changing the oligosaccharide side chains and making the amino acid substitutions in the receptor-binding site affected HA receptor-binding specificity. We constructed HK/97 HA mutants that had the two additional glycosylation sites (HK/97 HA.131/158+) as well as one or both of the amino acid substitutions at positions 138 and/or 227 (Fig. 3; 131/158+//A138S, 131/158+//S227N and 131/158+//A138S/S227N). HK/97 HA mutants with the glycosylation sites did not exhibit the haemadsorption phenotype shown by Ty/Ont HA when only one of the amino acid substitutions was introduced (HK/97 HA.131/158+//A138S and 131/158+//S227N). However, when both amino acid substitutions were made, the HK/97 HA with the additional glycosylation sites (HK/97 HA.131/158+//A138S/S227N) was adsorbed by chicken (to a limited extent probably due to suboptimal binding affinity) but not human RBCs, a haemadsorption phenotype similar to that of Ty/Ont HA. This mutant bound to human RBCs after sialidase treatment. Thus, we conclude that both glycosylation and the amino acid sequence in the receptor-binding site cooperatively determine receptor-binding specificity of HA.
Here we have shown a difference in the haemadsorption phenotype with human RBCs between the index H5N1 human isolate in 1997 and a virulent avian virus. This difference relates to the affinity of these two viruses for SA2,3Gal-containing receptors on human cells, and may be explained by a mechanism similar to that recently reported for H3N2 viruses (Nobusawa et al., 2003
). The H3N2 human viruses, isolated after 1992, do not agglutinate chicken RBCs and also have reduced binding activity to SA
2,6Gal-containing sialyloligosaccharides in MDCK cells. However, these viruses bind tightly to MDCK cells that have been desialylated and then resialylated with N-acetyl-D-neuraminyl-(
2,6)-D-galactopyranosyl-(
1,4)-N-acetyl-D-glucosamine, suggesting that the asialo portion of the sialyloligosaccharides may be responsible for receptor differentiation. Identification and characterization of SA
2,3Gal-containing receptors on human cells together with binding analysis of human and avian isolates to these molecules would further our understanding of the receptor specificity changes that occur when viruses are transmitted from birds to humans.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Claas, E. C., Osterhaus, A. D. M. E., van Beek, R., De Jong, J. C., Rimmelzwaan, G. F., Senne, D. A., Krauss, S., Shortridge, K. F. & Webster, R. G. (1998). Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472477.[CrossRef][Medline]
Connor, R. J., Kawaoka, Y., Webster, R. G. & Paulson, J. C. (1994). Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology 205, 1723.[CrossRef][Medline]
Couceiro, J. N. S. S., Paulson, J. C. & Baum, L. G. (1993). Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium: the role of the host cell in selection of hemagglutinin receptor specificity. Virus Res 29, 155165.[CrossRef][Medline]
Garcia, M., Crawford, J. M., Latimer, J. W., Rivera-Cruz, E. & Perdue, M. L. (1996). Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico. J Gen Virol 77, 14931504.[Abstract]
Ha, Y., Stevens, D. J., Skehel, J. J. & Wiley, D. C. (2001). X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proc Natl Acad Sci U S A 98, 1118111186.
Ha, Y., Stevens, D. J., Skehel, J. J. & Wiley, D. C. (2002). H5 avian and H9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes. EMBO J 21, 865875.
Hatta, M., Gao, P., Halfmann, P. & Kawaoka, Y. (2001). Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293, 18401842.
Horimoto, T. & Kawaoka, Y. (1994). Reverse genetics provides a direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus. J Virol 68, 31203128.[Abstract]
Horimoto, T., Fukuda, N., Iwatsuki-Horimoto, K. & 7 other authors (2004). Antigenic differences between H5N1 human influenza viruses isolated in 1997 and 2003. J Vet Med Sci (in press).
Ito, T., Suzuki, Y., Mitnaul, L., Vines, A., Kida, H. & Kawaoka, Y. (1997). Receptor specificity of influenza A viruses correlates with the agglutination of erythrocytes from different animal species. Virology 227, 493499.[CrossRef][Medline]
Ito, T., Couseiro, J. N. S. S., Kelm, S. & 8 other authors (1998). Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol 72, 73677373.
Kobasa, D., Rodgeres, M. E., Wells, K. & Kawaoka, Y. (1997). Neuraminidase hemadsorption activity, conserved in avian influenza A viruses, does not influence viral replication in ducks. J Virol 71, 67066713.[Abstract]
Matrosovich, M., Zhou, N., Kawaoka, Y. & Webster, R. G. (1999). The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J Virol 73, 11461155.
Matrosovich, M., Tuzikov, A., Bovin, N., Gambaryan, A., Klimov, A., Castrucci, M. R., Donatelli, I. & Kawaoka, Y. (2000). Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J Virol 74, 85028512.
Medeiros, R., Escriou, N., Naffakh, N., Manuguerra, J.-C. & van der Werf, S. (2001). Hemagglutinin residues of recent human A (H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes. Virology 289, 7485.[CrossRef][Medline]
Niwa, H., Yamamura, K. & Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193199.[CrossRef][Medline]
Nobusawa, E., Ishihara, H., Morishita, T., Sato, K. & Nakajima, K. (2000). Change in receptor-binding specificity of recent human influenza A viruses (H3N2): a single amino acid change in hemagglutinin altered its recognition of sialyloligosaccharides. Virology 278, 587596.[CrossRef][Medline]
Ohuchi, M., Feldmann, A., Ohuchi, R. & Klenk, H.-D. (1995). Neuraminidase is essential for fowl plague virus hemagglutinin to show hemagglutinating activity. Virology 212, 7783.[CrossRef][Medline]
Rogers, G. N. & D'Souza, B. L. (1989). Receptor binding properties of human and animal H1 influenza virus isolates. Virology 173, 317322.[Medline]
Seo, S. H., Hoffmann, E. & Webster, R. G. (2002). Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nat Med 8, 950954.[CrossRef][Medline]
Shortridge, K. F. (1995). The next pandemic influenza virus? Lancet 346, 12101212.[Medline]
Subbarao, K., Klimov, A., Katz, J. & 13 other authors (1998). Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279, 393396.
Weis, W., Brown, J. H., Cusack, S., Paulson, J. C., Skehel, J. J. & Wiley, D. C. (1988). Structure of the influenza virus hemagglutinin complexed with its receptor, sialic acid. Nature 333, 426431.[CrossRef][Medline]
Wright, P. F. & Webster, R. G. (2001). Orthomyxoviruses. In Fields Virology, 4th edn, pp. 15331579. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott -Williams & Wilkins.
Received 21 July 2003;
accepted 23 December 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |