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Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of α2-6 and α2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to α2-6- and α2-3-type receptors but retained substantial binding to specific O- and N-linked α2-3 glycans, including α2-3GalNAc and fucosylated α2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.
Influenza A viruses are generally isolated and propagated in embryonated chicken eggs or in cultures of cells of mammalian origin. Human influenza viruses were previously noted to acquire mutations in the hemagglutinin (HA) gene upon isolation and culture in the allantoic sac of embryonated chicken eggs (herein simply referred to as “eggs”) compared to the sequences of those isolated in mammalian cell substrates (herein referred to as “cells”) (29, 30, 44, 53, 58). These mutations resulted in amino acid substitutions that were found to mediate receptor specificity changes and improved viral replication efficiency in eggs (37). In general, cell-grown viruses are assumed to be more similar than their egg-grown counterparts to the viruses present in respiratory secretions (30, 56). Since their emergence in 1968, influenza A (H3N2) viruses have evolved and adapted to the human host while losing their ability to be efficiently isolated and replicate in eggs, particularly after 1992 (37, 42, 48). The rate of isolation of H3N2 clinical specimens after inoculation into eggs can be up to ~30 times lower than that in mammalian cell cultures, highlighting the strong selective pressure for the emergence of sequence variants (77).
Virtually all influenza vaccines for human use were licensed decades ago by national regulatory authorities, which used a product manufactured from influenza viruses isolated and propagated exclusively in eggs; therefore, cell culture isolates have been unacceptable for this purpose (41, 71). The antigen composition of influenza vaccines requires frequent updates (every 2 years, on average) to closely match their antigenic properties to the most prevalent circulating antigenic drift variant viruses (51). The limited availability of H3N2 viruses isolated in eggs has on one or more occasions delayed vaccine composition updates and may have reduced the efficacy of vaccination against new antigenically drifted viruses (3, 34, 37).
Entry of influenza viruses into host cells is mediated by HA, which binds to sialic acid containing glycoconjugates on the surface of epithelial cells in the upper respiratory tract (2, 13). The nature of the linkage between sialic acid and the vicinal sugar (usually galactose) varies in different host species and tissues and may therefore determine whether an influenza virus binds to and infects avian or human cells (40, 46, 59, 62, 72-75). Human influenza viruses preferentially bind to α2-6-linked sialic acids, and avian viruses predominantly bind to α2-3-linked sialic acids (59). Previous studies with chicken embryo chorioallantoic membranes revealed differential lectin binding, suggesting that α2-3-linked but not α2-6-linked sialosides are present on the epithelial cells (28). Human H3N2 viruses isolated in cell culture were reported to bind with a high affinity to α2-6-linked sialosides, while viruses isolated in eggs often had increased specificity for α2-3-linked sialosides (19, 20, 28). The functional classification of avian and mammalian influenza virus receptors is further complicated since in vitro and tissue-binding assays have led to new working hypotheses involving glycan chain length, topology, and the composition of the inner fragments of the carbohydrate chain as additional receptor specificity determinants (9, 17, 65, 66, 82). However, the significance of these in vitro properties remains unknown, since the structures of the natural sialosides on host cells that are used for infectious virus entry are undefined.
The techniques most widely used to study the interactions of the influenza virus with host cell receptors employ animal cells in various assay formats (36, 57, 59, 64, 69). To overcome the problems of cell-based techniques, new assays that rely on labeled sialyl-glycoproteins or polymeric sialoglycans have been developed (18). However, these assays are limited by having only a few glycans available in polymeric form and offer low throughput. In contrast, glycan microarrays can assess virus binding to multiple well-defined glycans simultaneously. Previous work with influenza live or β-propiolactone (BPL)-inactivated virions as well as recombinantly produced HAs revealed a good correlation with receptor specificity compared to that achieved by other methods of analysis (4, 11, 57, 58, 65-68).
Here we have compared paired isolates derived in eggs or cell cultures from the single clinical specimen to better understand their receptor binding specificity and its implications for vaccine production. We examined the differences in the sequences of the HAs between egg- and cell-grown isolates and analyzed their receptor binding profiles using glycan microarrays. Sequence analysis of the HA and glycan binding results revealed two distinct groups of viruses, with many egg isolates showing unexpectedly reduced levels of binding to α2-3 and α2-6 sialosides compared to the levels for the viruses isolated in mammalian cells. Furthermore, these studies highlighted that specific glycans may be important for H3N2 virus growth in eggs.
Clinical specimens (respiratory secretions) and virus isolates were obtained from the WHO Global Influenza Surveillance Network. Virus isolation in cell culture was performed by inoculation of clinical materials into primary monkey kidney cells or Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection), cultured in Dulbecco's modification of Eagle's medium with trypsin supplementation as described previously (76) (Table (Table1).1). Isolation in eggs was performed as described previously (30, 76). Approximately 10 median (50%) egg infectious doses (EID50s) of virus harvested from eggs were used for subsequent inoculation; the total number of passages was limited to the minimum required to obtain the necessary amounts of virus for analysis. The viruses and their passage histories are listed in Table Table1,1, along with abbreviations. Both cell- and egg-adapted WY03 viruses were generated by reverse genetics (10, 16, 27, 45) on the basis of HA sequences of the cell and egg isolates and a complete wild-type genome (GenBank accession number EU502435). The HA genes of all virus stocks used in this study were sequenced to detect the emergence of sequence variants (and quasispecies) during growth and amplification. Detection of variant sequence populations was based on the presence of discrete overlapping peaks in the capillary sequencer chromatogram. Subdominant viral populations with a relative abundance of ≥20% were detected with confidence by this method. High-growth reassortant viruses with the HA and neuraminidase genes of BR07, the M gene from A/Puerto Rico/8/34, and the remaining genes from either parent were derived as described previously (32).
The BR07 HA cell isolate and X-171B models were generated by homology modeling (61) using the structure of A/Aichi/2/1968 (Protein Data Bank accession number 2HMG) as the template.
Viruses were grown in either MDCK cells or embryonated chicken eggs, clarified by low-speed centrifugation, and inactivated by treatment with BPL (0.05%) for 3 days at 4°C under conditions that did not significantly alter the hemagglutinating titers (5). Inactivated virus-containing supernatant liquids were concentrated as described previously (52). Virus stocks were aliquoted and stored at −80°C. Virus concentrations were determined from the amount of HA using 0.5% (vol/vol) turkey red blood cells, since they contain both α2-3 and α2-6 sialoglycans (70). A pilot study was performed to determine the impact of BPL inactivation on the profile of glycans bound by A/Brisbane/10/2007 (H3N2) reassortant X-171B; live and inactivated viruses were analyzed in the same experiment, as described previously (11).
Glycan microarray slides were produced specifically for influenza research at the CDC using the Consortium for Functional Glycomics (CFG) glycan library (see Table S1 in the supplemental material for the glycans used for analyses in these experiments), as described previously (4). Virus preparations were thawed and diluted in phosphate-buffered saline (PBS) with 3% (wt/vol) bovine serum albumin (BSA) to an HA titer of 128, established to be optimal for glycan array analyses in this study, as described below. Virus suspensions were applied to the slides, and the slides were incubated in a closed container (at 4°C) subjected to gentle agitation for 1 h. Unbound virus was washed off with brief sequential rinses in PBS with 0.05% Tween 20 (PBS-T) and PBS. The slides were then immediately incubated with sheep serum hyperimmune to H3 HA (30 min); a biotinylated anti-sheep IgG antibody (30 min), and a streptavidin-Alexa Fluor 488 conjugate (30 min) (Invitrogen, CA), with brief PBS-T/PBS washes being performed after the incubations. After the final PBS-T/PBS washes, the slides were briefly washed in deionized water, dried by a gentle steam of air, and immediately subjected to imaging. Fluorescence intensities were detected using a ProScanArray HT apparatus (Perkin-Elmer). Image analyses were carried out using ImaGene 8 image analysis software (BioDiscovery, El Segundo, CA). For a simplified graphical representation, data were processed in the Microsoft Excel program to group similar sialoglycans together. Glycans that bound to the antibody-only control slide (see Fig. S1 in the supplemental material) were removed from the results. A/Brisbane/10/2007 (H3N2) was analyzed in the glycan microarray at 2-fold incremental HA titers between 16 and 128 to determine the range of virus concentrations that would yield optimal fluorescence signals (see Fig. S3 in the supplemental material).
We analyzed paired viruses isolated from nine clinical samples collected during 2006 and 2007 and propagated in either mammalian cells or embryonated eggs. One additional virus pair from a clinical specimen collected in 2003, WY03, was generated by reverse genetics, based on the available sequence information (see Table Table11 for all virus abbreviations) (16, 27, 45). Sequence analysis of the HAs from the paired isolates shows that the majority of amino acid changes in the egg-grown virus relative to the sequence of the cell-grown virus occur at 8 of the 16 positions that have previously been reported to be egg isolation-associated sites in older viruses, i.e., positions 138, 156, 168, 183, 186, 193, 194, and 219 (Table (Table2)2) (19, 24, 26, 30, 31, 38, 43, 44, 78, 83, 84). This study also revealed changes at an additional seven new positions (positions 86, 142, 190, 195, 198, 223 and 225) that have not previously been associated with H3N2 virus growth in eggs, although two of these amino acid substitutions (at positions 190 and 195) were found only as mixed populations (Fig. (Fig.1;1; Tables Tables22 and and3).3). Notably, amino acid substitutions at two of these positions (190 and 225) were previously reported in egg-grown H1N1 viruses (19, 54) (Fig. (Fig.11).
Comparison of cell and egg isolates revealed no consensus amino acid substitutions that could be used to predict how future viruses will be affected by isolation and growth in eggs. Indeed, the results revealed from 1 to 4 amino acid substitutions in HA1 of each egg-grown virus. Interestingly, the Gly186Val substitution was common to 6 of the 10 viruses studied (NY206, NJ06, PA07, WI07, WY03, and BR07), while the Leu194Pro/Met replacement was common to four viruses (FL06, NH06, NY306, and BR07) (Tables (Tables22 and and3).3). Of these two sites, variation at position 186 has been associated with egg propagation (19, 24, 26, 31, 38, 44, 78, 83) more frequently than variation at position 194 (26, 43). The HA1 sequences of the nine isolates from 2006 and 2007 differed from each other by 1 to 3 amino acids at six other positions not previously reported to be associated with egg isolation and propagation: positions 25, 50, 79, 83, 140, and 173 (Table (Table22).
To determine the receptor specificity of recent H3N2 viruses isolated in mammalian cells or in eggs, 10 paired egg and cell isolates were subjected to glycan microarray analysis. For egg-isolated viruses, only low-egg-passage-number viruses (i.e., passaged two to five times at a low multiplicity) were analyzed by the glycan microarray because very few further changes were observed at higher passage levels. To compare the viruses recovered in 2006 and 2007 to earlier isolates analyzed in studies described in the published literature (35), we included WY03 cell- and egg-like viruses generated from the available sequences by reverse genetics (Table (Table1).1). Pilot studies were conducted to evaluate the impact of inactivation with BPL and the virus concentration on glycan binding profiles, as described previously (11) (see Fig. S2 and S3 in the supplemental material). These studies showed that inactivation with BPL did not affect virus binding at the concentration established to be optimal (HA titer of 128) (see Fig. S2 and S3 in the supplemental material).
The glycan microarray results from this study revealed that certain glycans were ligands for most egg- and cell-derived H3N2 viruses analyzed. Glycan specificities shared by egg and cell-derived viruses were most likely present in the viruses in the original clinical specimen, since these two different host systems would not be expected to impose identical and parallel selective pressures. All viruses bound to a α2-6 sialylated tri-N-acetyllactosamine glycan in which the two proximal (reducing end) lactosamines are 1-3 fucosylated (Fig. (Fig.22 to 4; glycan 57 in the figures; Table Table4).4). This polylactosamine structure was reported to be a terminal sequence in the N-linked glycans of some mammalian cells (9). In addition, ~75% of the isolates bound to a structurally related long linear α2-6 sialylated di-N-acetyllactosamine (Fig. (Fig.22 to to4,4, glycan 56), although egg isolates of NH06, NY306, PA07, WI07, and BR07 did not bind. This glycan was also detected as a terminal sequence in N-glycans of cultured human bronchial epithelial cells (9). Several other structurally diverse glycans were also broadly recognized receptors: (i) most viruses bound α2-6 sialylated N,N′-diacetyllactosediamine (LacDiNAc) (glycan 55); the exceptions were cell and egg isolates of WI07 and egg isolates of NH06 and NY306; (ii) most viruses bound the α2-6 sialylated-sulfated N-acetyllactosamine structure (glycan 41); the exceptions were egg isolates of NY306, PA07, WI07, and BR07; and (iii) most viruses bound a sialyl Lewis x motif (glycans 29 and 30); the exceptions were cell and egg isolates of HO06 and the FL06 cell isolate. Glycans 41 and 55 have been detected in normal or inflamed human tissues (14, 15), whereas sialyl Lewis x glycans are present in the human respiratory tract (1, 23). As with some H3N2 viruses analyzed previously, the isolates in this study (except cell-derived WY03) bound poorly or not at all to α2-6 sialylated biantennary glycans, typically found on membrane glycoproteins (glycans 46 to 49) (35, 66). Collectively, these findings suggest that some H3N2 viruses in original clinical specimens may bind a more diverse set of sialoglycans than was previously reported.
The isolate pairs analyzed separated into distinct groups on the basis of their glycan binding profiles. The results for group 1 viruses, comprising FL06, HO06, and NH06, were characterized by the similar binding profiles for both their cell- and egg-derived isolates (Fig. Fig.2).2). Viruses had restricted binding to a core subset of linear glycans terminated with α2-6 sialyl N-acetyllactosamine (glycans 53 to 57), although GalNAc was tolerated in place of Gal (α2-6 sialylated LacDiNAc [glycan 55]). For the three viruses, limited α2-3 specificities were apparent, with good binding only to the N-linked α2-3 sialylated LacDiNAc (glycan 21) occurring (60). Comparing HA sequences between the cell and egg isolate pairs, HO06 revealed only one amino acid difference, an isoleucine at position 86 of the cell isolate and a leucine in the egg counterpart (Ile86Leu). NH06 had only a mixed sequence population at position 194 (Pro/Leu in cell-derived virus and Pro in egg-derived virus), while the two FL06 sequences revealed mixed populations at five residues in their HA1 sequences, which may explain the minor differences in their binding profiles.
Comparison by glycan microarray of the cell- and egg-grown viruses for the second group (group 2), comprising NY306, NJ06 NY206, PA07, WI07, WY03, and BR07 viruses, revealed a much more significant change in receptor specificity (Fig. (Fig.33 and and4).4). Cell-grown viruses revealed a much broader specificity for binding to many α2-3 and α2-6 glycans, as well as the α2-3/α2-6 disialoside and biantennary N-linked glycans (60-64) relative to that of group 1 cell isolates (Fig. (Fig.22 and and3).3). Notably, the binding of cell-derived NY206 to α2-3 glycans was relatively weaker than that by the other viruses in group 2. In addition, egg-isolated viruses generally revealed a significant reduction in α2-6 binding, although binding to one or more of the α2-6 sialyl N-acetyllactosamine motifs (glycans 53 to 57) was maintained, as seen in the group 1 viruses. The most dramatic reduction was seen for the 2007 Wisconsin egg isolate (WI07), which bound weakly to all α2-6 sialylglycans. Binding to α2-3 avian-type receptors was also reduced for all of these group 2 egg-derived isolates. Only fucosylated α2-3 glycans (glycans 28 to 33) were maintained or enhanced upon egg isolation/propagation (Fig. (Fig.3).3). Despite these dramatic differences in binding profiles, comparison of the HA1 sequences revealed that at the protein level, the egg- and cell-derived isolate pairs differed by only 2 amino acid substitutions (WY03, NY306, WI07), 3 substitutions (PA07), or 4 substitutions (NJ06, NY206, BR07) (Table (Table22 and and3).3). In five out of the six group 2 viruses shown in Table Table2,2, a Gly186Val substitution was introduced during egg isolation and propagation, together with a second substitution in or near the loop at position 220 (220 loop; NJ06, NY206, PA07, WI07, WY03). The only egg-derived virus not to conform to this observation was NY306, in which a Leu194Pro substitution was introduced within the alpha helix that sits atop the receptor binding site (RBS), and an Ile223Val substitution was introduced in the 220 loop. Interestingly, in three of the previous five viruses (NY206, NJ06, and WI07), the 220-loop substitutions introduced bulkier and/or hydrophobic residues when they were adapted to eggs. The remaining two viruses, WY03 and PA07, had changes at positions 226 and 225, respectively, positions previously identified in modifying receptor specificity in H3 and H1 viruses (39, 48, 66, 83). The structural basis for group 1 or 2 binding profiles remains to be determined; these data do not allow a direct linkage between HA sequence and receptor function, precluding attempts to predict receptor binding profiles on the basis of amino acid sequences.
The WY03 virus (also in group 2) was of particular interest since the sialoside binding pattern of the egg variant-derived vaccine reassortant has already been published (35). The egg-derived isolate showed binding comparable to that published previously. However, the paired MDCK cell culture isolate from the same patient specimen exhibited the broad specificity characteristic of other group 2 viruses isolated in MDCK cell culture (Fig. (Fig.3).3). In addition to α2-3 and α2-6 binding, WY03 egg-derived virus also bound weakly to α2-8 glycans (glycans 75, 76, and 79; see Fig. Fig.33 and reference 35), in contrast to the virus from MDCK cell cultures. This specificity was also previously reported for an egg-adapted NWS-Memphis/31/1998 (H3N2) reassortant virus (81), raising the possibility that 8′-polysialic acid receptor binding by influenza A (H3N2) viruses may result from isolation in eggs.
We also compared the BR07 cell isolate to the egg counterpart and to three clonal populations derived from the latter by limiting-dilution passage in eggs to generate high-growth reassortants for vaccine production (32) (Table (Table1).1). Compared to the cell isolate, biologically cloned egg-derived viruses had one to three changes in the HA. X-171 had three substitutions (Gly186Val, Ala198 deletion [del], and Ile226Ser), X-171A had a single change (Leu194Pro), and X-171B also had three substitutions (Gly186Val, Ala198 deletion, and Ser219Tyr) in their HA1 (Table (Table33).
Sequence analysis of the uncloned egg-isolated BR07 virus after five egg passages revealed the persistence of a heterogeneous virus population with variable residues at positions 186 (Gly/Val), 190 (Asp/Asn), 194 (Leu/Pro), and 198 (Ala/deletion) in the HA (Table (Table3).3). In agreement with these observed substitutions, glycan microarray analysis of the egg- and cell-grown BR07 viruses revealed characteristic group 2 profiles (Fig. (Fig.4).4). Since the heterogeneous populations in the BR07 egg-grown virus that was analyzed included substitutions identified in the X-171, X-171A, and X-171B reassortants, it was not surprising to see the binding profiles for these viruses yielding results similar to those for the uncloned original egg isolate (Fig. (Fig.4).4). The most unexpected result, though, appeared with the high-growth X-171B reassortant. Glycan microarray analysis of X-171B revealed a highly restricted pattern of binding to only eight glycans on the microarray: N-linked α2-3 sialylated LacDiNAc (glycan 21), sialyl Lewis x motifs (glycans 29 to 33), α2-6 sialylated LacDiNAc (glycan 55), and α2-6 sialylated tri-N-acetyllactosamine (glycan 57) (Fig. (Fig.4;4; Table Table4).4). This short list of candidate sialoglycans may help identify the receptor(s) essential for efficient virus propagation in chicken embryos.
Successful replication of influenza viruses in eggs was first reported in 1940 (6). The allantoic sacs of embryonated eggs became an ideal substrate for influenza vaccines because they supported high antigen yields. The vast majority of current influenza vaccines are also produced in eggs from viruses propagated exclusively in eggs because vaccines produced in cell culture systems have not yet been licensed in most countries (22, 33, 50). Although the egg allantoic sac has been used successfully to produce influenza vaccines for 60 years, recent human H3N2 viruses have largely lost the ability to replicate in this host unless they acquire certain mutations in the HA (48, 80). This would not become a major problem if a vaccine virus seed could be used for many years after its derivation in eggs. However, rapid antigenic drift mandates frequent updates in influenza vaccine composition, which requires derivation of new virus seeds in eggs within a short period of time (21).
Host selection of influenza A virus mutants has been known for over 65 years (7, 8, 12, 55), but we have not yet fully characterized the role of egg propagation on amino acid changes and virus receptor specificity. Cell-grown viruses have been demonstrated to accumulate fewer mutations than viruses present in the respiratory tract (29, 30, 53, 58) and thus are more representative of the wild-type virus circulating in the human population.
Previous studies have identified 16 positions at which substitutions emerged in the HA of H3N2 viruses isolated in eggs. However, the majority of these studies reported using viruses isolated between 1986 and 1990 (19, 24, 26, 30, 31, 44, 56, 83), while one studied a single 1997 virus (43) and two studies looked at a A/Fujian/2002-like virus (38, 78). Our studies with 9 pairs of viruses isolated in 2006 and 2007 identified substitutions at 8 of these 16 previously reported positions, and all were close to the RBS (Fig. (Fig.1).1). Formal demonstration of their role in viral replication in the egg host system would require detailed analysis of isogenic mutant viruses. However, their potential significance for replication in eggs can be inferred from the nonrandom distribution of the 16 changes in the HA (Table (Table2)2) with regard to spatial and population diversity considerations. First, the majority of the variation occurred at only eight amino acid positions, all of which are vicinal to the receptor binding site in the HA1. Second, most changes involved the 16 amino acids previously reported to vary in relation to isolation in eggs (19, 24, 26, 30, 31, 38, 43, 44, 56, 78, 83).
On the basis of their binding profiles to glycan microarrays, the virus pairs studied could be divided into two groups. Group 1 HAs had only minimal differences in glycan microarray binding, and this was perhaps reflected in the somewhat reduced number of substitutions observed between the cell- and egg-derived HAs. Glycan array analysis of these cell isolates revealed that they already possessed a restricted sialoside binding profile. This property was associated with specific amino acids at critical positions; e.g., cell-derived NH06 had a mixed population at position 194 (Leu/Pro), while the HO06 cell-derived isolate already had a Leu at position 183, previously reported to be egg-selected (38). Group 2 virus isolates from eggs, however, had more dramatic changes with respect to their glycan binding profiles; receptor recognition was restricted to a smaller subset of glycans compared to the size of the subset in their cell grown counterparts. These changes suggested that egg isolation was frequently associated with a Gly186Val substitution in combination with a substitution in the 220 loop of HA. The NY306 egg isolate was the exception, having a Leu194Pro substitution besides the 220-loop modification.
Substitutions were also observed at seven additional sites that had not been previously reported for H3N2 viruses isolated in eggs (colored blue in Fig. Fig.1).1). Five of the seven sites (190, 195, 198, 223, and 225) are within or proximal to the RBS. Position 190 substitutions have been associated with the replication of H1N1 viruses in eggs (39, 66), while position 198 was deleted in one of the BR07 clones (discussed below). Residues 223 and 225 are in the 220 loop, which was previously reported to be subject to changes during growth in eggs (44). Collectively, changes at these five positions merit further studies to determine their role in efficient viral replication in the egg host system.
Our results from the BR07 egg-isolated high-growth reassortants can shed light on virus-host interactions in this system. The X-171A clone had a single change (Leu194Pro), while the X-171B clone had two amino acid substitutions and one deletion (Leu186Pro, Ala198del, and Ser219Tyr) (Table (Table3).3). The uncloned BR07 isolated in eggs revealed heterogeneous populations at three of the four positions (Gly/Val at position 186, Leu/Pro at position 194, Ala/del at position 198), confirming the presence of most of these changes prior to selection of clonal reassortant viruses. Asp/Asn at 190 was the only change not seen in the cloned reassortants. Interestingly, two of the three changes observed in the high-growth reassortant (X-171B) were present in the egg passage 5 virus and predominated in the passage 9 virus (data not shown), suggesting that the Gly186Val/Ala198del combination is beneficial for growth in eggs. These two changes, in combination with an additional Ser219Tyr substitution (near the 220 loop), resulted in a dramatic switch in receptor specificity with a highly restricted binding profile (Fig. (Fig.4).4). Furthermore, a high-growth virus with a deletion in the HA (Ala198del) was obtained in eggs, despite the scarcity of trinucleotide deletions relative to single base substitutions in the original virus population, suggesting that the Ala198del/Gly186Val combination imparted a replication advantage in eggs. Antigenic analyses of the cell and egg isolates by hemagglutination inhibition tests using a panel of ferret antisera to A/Brisbane/10/2007 wild-type virus and each of its reassortants failed to reveal a ≥8-fold and reciprocal titer differences, although a partial (nonreciprocal) ≤8-fold difference was noted in these studies (data not shown).
Disruption of the helix at position 190 resulting from the introduction of a Leu194Pro substitution in BR07 (as well as in NH06 and NY306) appears to be correlated with a glycan binding profile similar to that of the X-171B high-yield reassortant (Fig. (Fig.33 and and4).4). The same appears to happen for the deletion at position 198, although its true effect cannot be determined due to the additional substitutions at positions 186, 219, and 226, which may also play a role (as seen with the other group 2 viruses). Position 186 and the 220-loop substitutions all introduce bulkier and/or hydrophobic residues to the left side of the RBS (Fig. (Fig.1).1). Comparing glycan microarray binding of X-171 and X-171B, which differ only in the position of the substitution on the 220 loop (Ile226Ser in X-171 and Ser219Tyr in X-171B), suggests that position 219 is the critical residue responsible for a more restrictive binding profile. Modeling a sialyl Lewis x glycan (Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ) into a hypothetical BR07 binding pocket indicates that the fucose could directly interact with the hydroxyl of the phenolic side chain of the Tyr and might therefore selectively stabilize these glycans (Fig. (Fig.5).5). In contrast, an Ile226Ser substitution introduces a potential hydrogen bond to the O-4 of galactose, which is less selective.
The narrow receptor specificity of the high-growth X-171B reassortant raises the possibility that these glycans might be present and used by the virus during egg growth: α2-3 sialylated LacDiNAc (glycan 21), sialyl Lewis x (glycans 29 to 33), α2-6 sialyl LacDiNac (glycan 55), α2-6 sialyl fucosyl tri-lactosamine (glycan 57), and sialylated N-glycolyl Lewis x (glycan 68). However, to date, these structures have not been identified as terminal sequences on N-linked glycans of either embryonated chicken egg chorioallantoic or amniotic cells (63), though neutral nonsialylated glycans containing LacDiNAc as well as fucosylated N-acetyllactosamine have been described in the N-glycan structures of a chicken eggshell protein, ovocleidin-116 (47), indicating that chickens have the biosynthetic capability to make the corresponding sialylated structures.
Kumari et al. recently analyzed MDCK cell-grown viruses isolated between 2003 and 2005 and found restricted specificity to only α2-6 glycans (35). Except for WY03, all the virus pairs presented in this study are isolated from 2006 onwards and revealed a much broader specificity. A possible reason for this difference may be due to a Ser193Phe substitution that emerged in 2005 and that is currently the dominant residue in circulating strains. Analysis of the HA sequences from the study of Kumari et al. reveals a serine at this position. Position 193 is in the alpha helix that sits atop the RBS, and the bulky aromatic side chain is predicted to extend over the receptor pocket. This, in conjunction with substitutions (Ser189Asn and Ser227Pro) on the left side of the RBS in regions of importance for egg adaptation, appeared in viruses from 2004 onwards and may contribute to the increased α2-3 binding observed in this study. Interestingly, Gulati et al. reported that cell-derived Brisbane/10/07-like viruses bound to very few sialoglycans on the array, primarily α2-6 sialyl-polylactosamines (25). The reason why their findings are so different from those for the viruses analyzed here is unknown. An analysis of the virus dilution of both cell- and egg-grown A/Brisbane/10/2007 (H3N2) virus at 2-fold incremental HA titers between 32 and 512 yielded an overall reduction in overall glycan signals rather than enrichment of the single long polylactosamine reported previously (see Fig. S3 in the supplemental material), suggesting that a drastic difference in the HA titers used between these different laboratories is not the reason. The rapid evolution of the receptor binding preferences of H3N2 antigenic variants that emerged between 2003 and 2007 (Fujian/410-like, Wisconsin/67-like, California/7-like, and Brisbane/10-like viruses) highlights the need to reevaluate functional predictions with any future variant viruses of interest (25, 35).
Compared to viruses isolated in MDCK cell culture, egg isolates from 2006 and 2007 can have both modestly and dramatically different glycan microarray profiles. In addition, substitutions at different positions, such as 194 (X-171A) and 186/198 (X-171B), appear to produce similar binding profiles, thus illustrating the complexity of the system that we are attempting to understand. Williams and Robertson (79) reported that substitutions in the vicinity of the RBS appear to be responsible not only for modulating attachment to receptors but also for triggering virus penetration into the target cells. The mutations acquired by group 1 egg-derived isolates (and some of those of group 2-derived egg isolates) may modulate postbinding events mediating endocytosis and membrane fusion.
Although early studies showed that the sequences of HA genes of H3N2 viruses isolated in cell culture are generally identical to those sequenced directly from nasal secretions of patients, recently circulating viruses may also undergo host selection during isolation in mammalian cells (30, 49). Isolation of H3N2 viruses in MDCK cells has been progressively more difficult in recent years (49). This obstacle has been overcome by utilizing a transfectant MDCK cell line expressing higher levels of α2-6 sialosides, suggesting that the paucity of these receptors in regular MDCK cells may consequently exert selective pressure for selection of viruses with enhanced binding of α2-3 sialylated glycans. Most group 2 viruses isolated in cells show moderate to high levels of α2-3 sialoside specificity. Viruses in this group with strong α2-3 binding specificity also bind to α2-3 linked N-glycans as well as mixed 2-3/2-6 biantennary glycans (glycans 60 and 61, respectively). In contrast, this is not seen in many of the egg isolates. This raises the possibility that the α2-3 specificity seen in the MDCK cell-grown virus could reflect selection of viruses with increased specificity for α2-3 N-linked glycans to compensate for the low level of α2-6 N-linked glycans. Thus, the differences in glycan specificity seen between the egg- and cell-grown viruses could result from selective pressures exerted by both laboratory hosts.
These results also provide a snapshot of the receptor specificity of human H3N2 influenza viruses circulating in 2006 and 2007. Two long α2-6 sialylated glycans (glycans 56 and 57) were the ligands preferred by most viruses, a finding consistent with that from a previous report of a study that used recombinant HA (66). Three trisaccharides less frequently considered influenza virus receptors were the ligands widely used by these viruses. Two were α2-6 sialylated LacDiNAc and sulfated N-acetyllactosamine (glycans 55 and 41, respectively). Interestingly, the sialyl Lewis x glycan, whose presence in airway epithelial cells has been well documented (1, 23), was also an excellent ligand for many of the viruses in this panel, suggesting that α2-3-linked sialic acids should not be excluded as potential receptors for human influenza viruses. The consensus glycans for the 2006 and 2007 viruses includes the three previously reported ligands for the recombinant HA of A/Moscow/10/1999 (H3N2) virus (66): sulfated α2-6 sialyl N-acetyllactosamine (glycan 41), α2-6 sialyl di-N-acetyllactosamine (glycan 56), and α2-6 sialyl fucosylated tri-N-acetyllactosamine (glycan 57), indicating substantial conservation of receptor specificity (Table (Table4).4). The use of glycan arrays to analyze the receptor specificity of influenza viruses promises to advance our understanding of a very complex and dynamic virus-host interface. However, the body of information on influenza virus receptor specificity derived from glycan array analyses is still insufficient to interpret the general significance of the data.
Eggs will remain the most economical and practical vaccine production substrate until a cell-based alternative is fully established. Understanding how receptor binding is evolving in human H3N2 viruses circulating globally may explain the basis of poor replication in eggs and the significance of more radical structural changes, such as the Ala198 deletion or Leu194Pro substitution by BR07. Such changes may affect secondary structures in the HA and thus could impact the antigenicity of site B. Structural analysis of these changes will help to clarify their effects in both regards. Further studies will establish the feasibility of analyzing the receptor binding profiles of cell isolates to identify those that match a previously determined glycan binding profile correlated with permissive growth in eggs. As shown with group 1 viruses, the original sequences in certain clinical specimens may be less prone to the type of changes acquired by the HA1 of BR07 (group 2) to generate the high-growth reassortant in eggs. Such viruses would reduce the risk of potential antigenic changes in seed viruses selected for vaccine production.
We thank the WHO Global Influenza Surveillance Network for providing influenza virus isolates and Ian Barr for the viruses isolated in eggs for reassortant production. The glycan microarray data presented here will be made available online through the Consortium for Functional Glycomics website upon publication (www.functionalglycomics.org).
The glycan microarray was produced for the Centers for Disease Control and Prevention by using a glycan library generously provided by the Consortium for Functional Glycomics, funded by National Institute of General Medical Sciences grant GM62116.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry.
Published ahead of print on 2 June 2010.
†Supplemental material for this article may be found at http://jvi.asm.org/.