Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes

To identify the molecular determinants contributing to the inability of rec ent human influenza A(H3N2) viruses to agglutinate chicken erythrocytes, ph enotypic revertants were selected upon passage in eggs or MDCK cells. The L eu194Ile or Val226Ile substitutions were detected in their hemagglutinin (H A) sequence concomitantly with the phenotypic reversion, Remarkably, as lit tle as 3.5% of variants bearing a Val226Ile substitution was found to confe r the ability to agglutinate chicken erythrocytes to the virus population. Hemadsorption assays following transient expression of mutated HA proteins showed that the successive Gln226 --> Leu - Ile --> Val changes observed on natural isolates resulted in a progressive loss of the ability of the HA t o bind chicken erythrocytes. The Val226Ile change maintained the preference of the HA for SA alpha2,6Gal over SA alpha2,3Gal and enhanced binding of t he HA to alpha2,6Gal receptors present on chicken erythrocytes. In contrast , simultaneous Ser193Arg and Leu194Ile substitutions that were found to con fer the ability to agglutinate sheep erythrocytes increased the affinity of the HA for SAa2,3Gal. (C) 2001 Academic Press.