J. Okada and others Fig. 1. Amino acid sequences of the seven antigenic sites in template and chimaeric HAs constructed in this study. (a) HAs of five vaccine strains, Aichi/68 (Aic68), Yamanashi/77 (Yam77), Fukuoka/85 (Fuk85), Sydney/97 (Syd97), Wyoming/2003 (Wyo03), were used as starting materials. The deduced amino acid sequences showed discrepancies with the reported data (Okada et al., 2010) at six residues, which are highlighted in grey. Since we used these plasmids for the expression of HA the actual sequence determined is indicated in this figure. The name of the chimaeric HA and the amino acids that replaced the original ones are indicated under the template HA. Bars indicate the same amino acids as those of Aichi/68. (b) Location of the amino acids replaced in the chimaeric HA is indicated in the 3D structure of HA by using the examples of the Sydney/97 strain. The amino acids that formed a receptor-binding site were marked in grey. The amino acids replaced in the chimaera were marked as follows: site A, purple (121A), orange (131A) or red (142A); B1, blue; B2, light blue; C1, green; C2, light green; D, pink; E, yellow. et al., 2010) were divided into three sets. The first set consisted of 11 clones (classified into groups 1–3) that bound to the 1968–1973 strains. The second set consisted of four clones (classified into groups 4 and 5) that bound to the 1977–1999 strains and 54 clones (classified into groups 6–19) that bound to the 1977–1993 strains. The third set consisted of 14 clones (classified into groups 20 and 21) that bound to the 1993–2003 strains and 15 clones (classified into groups 23–27 and 29) that bound to the 1997–2003 strains. Fig. 2. Attachment of reticulocytes to the cells expressing wild-type (wt) or chimaeric HAs of Aichi/68 influenza strain. 293T cells were transfected with plasmid containing wt or chimaeric HA genes of the Aichi/68 stain and incubated with guinea-pig red blood cells. As a control, plasmid without the HA gene was used. 328 Journal of General Virology 92