Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection

Human influenza viruses are more efficiently isolated by inoculating patient samples into the amniotic rather than the allantoic cavity of embryonated chicken eggs, This type of cultivation selects virus variants with mutations around the hemagglutinin (HA) receptor binding site. To understand the molecular basis of these phenomena, we investigated the abundances of sialic acid (SA) linked to galactose (Gal) by the alpha-2,3 linkage (SA alpha 2,3Gal) and SA alpha 2,6Gal in egg amniotic and allantoic cells and in Madin-Darby canine kidney (MDCK) cells, Using SA-Gal linkage-specific lectins (Maackia amurensis agglutinin specific for SA alpha 2,6Gal and Sambucus nigra agglutinin specific for SA alpha 2,3Gal), we found SA alpha 2,3Gal in both allantoic and amniotic cells and SA alpha 2,6Gal in only the amniotic cells, MDCK; cells contained both linkages, To investigate how this difference in abundances of SA alpha 2,3Gal and SA alpha 2,6Gal in allantoic and amniotic cells affects the appearance of host cell variants in eggs, we determined the receptor specificities and HA amino acid sequences of two different patient viruses which were isolated and passaged in the amnion or in the allantois and which were compared with MDCK cell grown viruses, We found that the viruses maintained high SA alpha 2,6Gal specificities when grown in MDCK cells or following up to two amniotic passages; however, further passages in either the amnion or allantois resulted in the acquisition of, or a complete shift to, SA alpha 2,3Gal specificity, depending on the virus strain. This change in receptor specificity was accompanied by the appearance of variants in the population with Leu-to Gln mutations at position 226 in their HA, These findings suggest that lack of SA alpha 2,6Gal linkages in the allantois of chicken eggs is a selective pressure for the appearance of host cell variants with altered receptor specificities and amino acid changes at position 226.