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

Abstract

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 ␣-2,3 linkage (SA␣2,3Gal) and SA␣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␣2,6Gal and Sambucus nigra agglutinin specific for SA␣2,3Gal), we found SA␣2,3Gal in both allantoic and amniotic cells and SA␣2,6Gal in only the amniotic cells. MDCK cells contained both linkages. To investigate how this difference in abundances of SA␣2,3Gal and SA␣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␣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␣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␣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.

The presence of viral subpopulations in an infectious bronchitis virus vaccine with differing pathogenicity – A preliminary study

“…The apparent reduced ability of VicS-del to infect host cells does not necessarily indicate a loss of pathogenicity, but could indicate an altered capacity to bind to different cell receptors. It has been suggested that changes near the receptor binding domain of the S1 protein could modify the affinity of coronaviruses for different cell surface glycoproteins [57][58][59]. Potentially, the variations found in the S1 gene might have reduced the ability of VicS-del to infect respiratory tract cells, but not its ability to infect the cells of the chorioallantoic membrane (CAM) (where replication of IBV initially occurs in embryonated chicken eggs).…”

Madin‐Darby canine kidney cell sialic acid receptor modulation induced by culture medium conditions: Implications for the isolation of influenza A virus

“…[7][8][9][10][11][12][13] Madin-Darby canine kidney (MDCK) cell lines are widely available, easily amplify in culture, and are commonly used for isolating IAVs from a variety of species. 12,14 Previous studies indicated that MDCK cells cultured in the presence of fetal bovine serum (FBS) express both α-2,6-linked and α-2,3-linked SA receptor types and showed that some cells co-express both receptors. 9,14 In contrast, another study found that 98% of MDCK cells adapted to serum-free media (SFM) expressed only α-2,6linked SA receptors, with 2% of cells expressing both α-2,3-and α-2,6-linked SA receptors.…”

“…12,14 Previous studies indicated that MDCK cells cultured in the presence of fetal bovine serum (FBS) express both α-2,6-linked and α-2,3-linked SA receptor types and showed that some cells co-express both receptors. 9,14 In contrast, another study found that 98% of MDCK cells adapted to serum-free media (SFM) expressed only α-2,6linked SA receptors, with 2% of cells expressing both α-2,3-and α-2,6-linked SA receptors. 15 However, detailed characterization of SFM-adapted MDCK cells is needed because variations in SFM preparations may cause the cells to express different amounts of each SA receptor, altering IAV culture success.…”

The infection of primary avian tracheal epithelial cells with infectious bronchitis virus

“…It has been shown that serial passage using chicken embryos enforces the selection of adapted viruses and inevitably alters the genetic codes of the primary viral isolates, especially for genes involved in cell adsorption and viral replication [8,9]. The environment of the viral replication may also selectively modify the original cell tropism and pathogenesis of the isolated virus [15,25]. It has been shown that a serially embryo-passaged of infectious bronchitis viruses (IBV) M41 strain exhibited significantly attenuated pathogenesis within the oviduct compared to the parental strain [7].…”

“…In addition, egg-mediated epitope alterations in amplified isolates might attenuate the protective efficiency of these viruses when used to vaccinate against the challenge of a wild-type virulent strain [16,24]. For instance, after egg adaption, human influenza A virus, originally showing a2-6 sialic acid tropism only, exhibits increased hemagglutinin (HA) binding affinity to a2-3 sialic acid-containing gangliosides due to amino acid substitutions in the vicinity of the receptor binding site of the HA protein [8,15]. For primary viral isolates, providing natural host cells for replication might eliminate the selection pressure of an exotic growth environment and avert the alteration of cell tropism and consequent genomic mutations.…”