[The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses].

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

Immune Response and Protective Efficacy of Inactivated and Live Influenza Vaccines Against Homologous and Heterosubtypic Challenge.

Inactivated (whole-virion, split, subunit, and adjuvanted) vaccines and live attenuated vaccine were tested in parallel to compare their immunogenicity and protective efficacy. Homologous and heterosubtypic protection against the challenge with influenza H5N1 and H1N1 viruses in a mouse model were studied. Single immunization with live or inactivated whole-virion H5N1 vaccine elicited a high level of serum antibodies and provided complete protection against the challenge with the lethal A/Chicken/Kurgan/3/05 (H5N1) virus, whereas application of a single dose of the split vaccine was much less effective. Adjuvants increased the antibody levels. Addition of the Iso-SANP adjuvant to the split vaccine led to a paradoxical outcome: it increased the antibody levels but reduced the protective effect of the vaccine. All tested adjuvants shifted the ratio between IgG1 and IgG2a antibodies. Immunization with any of the tested heterosubtypic live viruses provided partial protection against the H5N1 challenge and significantly reduced mouse mortality, while inactivated H1N1 vaccine offered no protection at all. More severe course of illness and earlier death were observed in mice after immunization with adjuvanted subunit vaccines followed by the challenge with the heterosubtypic virus compared to challenged unvaccinated animals.

The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses.

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

The Effect of a Lipopolysaccharide from Rhodobacter capsulatus PG on Inflammation Caused by Various Influenza Strains.

The development of a specific inflammation in mice that had been infected by two influenza virus strains, A/chicken/Kurgan/5/2005 (H5N1) and A/Hamburg/2009 MA (H1N1), was studied. We investigated the effect of a non-toxic lipopolysaccharide from Rhodobacter capsulatus PG on the survival and body weight of the mice, production of IgG antibodies, and the induction of pro- and anti-inflammatory cytokines in blood serum. The administration of the R. capsulatus PG lipopolysaccharide was shown to induce interferon-ß synthesis, both in healthy and influenza A virus-infected mice, and to promote production of antiviral antibodies in the blood of the influenza-infected animals.

Changes in the Receptor-Binding Properties of H3N2 Viruses during Long-Term Circulation in Humans.

It was previously shown that hemagglutinin residues Thr155, Glu158, and Ser228 are crucial for the recognition of Neu5Gc. In this study, we demonstrated that the ability to bind the Neu5Gc-terminated receptor is related to the amino acid 145: viruses of years 1972-1999 with Lys145 bind to the receptor, whereas viruses with Asn145 do not. Sporadic appearance and disappearance of the ability to bind Neu5Gc oligosaccharides and the absence of Neu5Gc in the composition of human glycoconjugates indicate the non-adaptive nature of this ability. It was previously shown that unlike H1N1 viruses, H3N2 viruses of years 1968-1989 did not distinguish between Neu5Acα2-6Galß1-4Glc (6'SL) and Neu5Acα2-6Galß1-4GlcNAc (6'SLN). H3N2 viruses isolated after 1993 have acquired the ability to distinguish between 6'SL and 6'SLN, similarly to H1N1 viruses. We found that the affinity for 6'SLN has gradually increased from 1992 to 2003. After 2003, the viruses lost the ability to bind a number of sialosides, including 6'SL, that were good receptors for earlier H3N2 viruses, and retained high affinity for 6'SLN only, which correlated with the acquisition of new glycosylation sites at positions 122, 133, and 144, as well as Glu190Asp and Gly225Asp substitutions, in hemagglutinin. These substitutions are also responsible for the receptor-binding phenotype of human H1N1 viruses. We conclude that the convergent evolution of the receptor specificity of the H1N1 and H3N2 viruses indicates that 6'SLN is the optimal natural human receptor for influenza viruses.

[Mutations in Hemagglutinin and Polymerase Alter the Virulence of Pandemic A(H1N1) Influenza Virus].

To study the pathogenicity factors of the pandemic A(H1N1) influenza virus, a number of mutant variants of the A/Hamburg/5/2009 (H1N1)pdm09 strain were obtained through passage in chicken embryos, mouse lungs, and MDCK cell culture. After 17 lung-to-lung passages of the A/Hamburg/5/2009 in mice, the minimum lethal dose of the derived variant decreased by five orders of magnitude compared to that of the parental virus. This variant differed from the original virus by nine amino acid residues in the following viral proteins: hemagglutinin (HA), neuraminidase (NA), and components of the polymerase complex. Additional passaging of the intermediate variants and cloning made it possible to obtain pairs of strains that differed by a single amino acid substitution. Comparative analysis of replicative activity, receptor specificity, and virulence of these variants revealed two mechanisms responsible for increased pathogenicity of the virus for mice. Thus, (1) substitutions in HA (Asp225Gly or Gln226Arg) and compensatory mutation decreasing the charge of HA (Lys123Asn, Lys157Asn, Gly158Glu, Asn159Asp, or Lys212Met) altered viral receptor-binding specificity and restored the functional balance between HA and NA; (2) Phe35Leu substitution in the PA protein increased viral polymerase activity.

[Three Mutations in the Stalk Region of Hemagglutinin Affect the pH of Fusion and Pathogenicity of H5N1 Influenza Virus].

Previously, an attenuated variant Ku/at was obtained from the highly pathogenic avian influenza virus A/chicken/Kurgan/3/2005 (H5N1) by a reverse selection method aimed at increasing the virus resistance to a proteolytic cleavage and acidic pH values. In the Ku/at, 10 mutations in proteins PB2, PB1, HA, NA, and NS1 occurred. In comparison with the parental strain, the pH of the conformational transition of the viral glycoprotein hemagglutinin (HA) and virulence for mice and chickens have decreased in an attenuated variant. The purpose of this work is to clarify the role of three mutations in the stalk region of HA: Asp54Asn in HA1 and Val48Ile and Lys131Thr in HA2 (H3 HA numbering). To attain these ends, analogous substitutions were introduced into HA with a deleted polybasic cleavage site (important for pathogenicity) of the recombinant A/Vietnam/1203/04-PR8/CDC-RG (H5N1) virus, and so we created the VN3x-PR variant. Viruses VN3x-PR and Ku/at with the same three mutations, but different proteolytic cleavage sites in HA, as well as the corresponding initial viruses, were tested for pathogenicity in mice and in the erythrocyte hemolysis test. Compared with the parental strains, the virulence of their mutant variants in the case of intranasal infection of BALB/c mice decreased by 4-5 orders of magnitude, and the pH of the conformational transition of HA decreased from 5.70-5.80 to 5.25-5.30, which is typical for low pathogenic natural isolates. Thus, as a result of the study, the attenuating role of these three mutations in HA has been proved, a correlation was established between the pH value of the HA conformational transition and the virulence of H5N1 influenza viruses, and it was shown that the polybasic cleavage site of the H5 HA does not always determine high pathogenicity of the virus.

[Changes in the phenotypic properties of highly pathogenic influenza A virus of H5N1 subtype induced by N186I and N186T point mutations in hemagglutinin].

The change in the phenotypic properties resulting from amino acid substitutions in the hemagglutinin (HA) molecule is an important link in the evolutionary process of influenza viruses. It is believed to be one of the mechanisms of the emergence of highly pathogenic strains of influenza A viruses, including subtype H5N1. Using the site-directed mutagenesis, we introduced mutations in the HA gene of the H5N1 subtype of influenza A virus. The obtained virus variants were analyzed and compared using the following parameters: optimal pH of conformational transition (according to the results of the hemolysis test), specificity of receptor binding (using a set of synthetic analogues of cell surface sialooligosaccharides), thermoresistance (heat-dependent reduction of hemagglutinin activity), virulence in mice, and the kinetics of replication in chicken embryos, and reproductive activity at different temperatures (RCT-based). N186I and N186T mutations in the HA protein increased the virulence of the original virus in mice. These mutations accelerated virus replication in the early stages of infection in chicken embryos and increased the level of replication at late stages. In addition, compared to the original virus, the mutant variants replicated more efficiently at lower temperatures. The obtained data clearly prove the effect of amino acid substitutions at the 186 position of HA on phenotypic properties of the H5N1 subtype of influenza A.

Immunization with live nonpathogenic H5N3 duck influenza virus protects chickens against highly pathogenic H5N1 virus.

Development of an effective, broadly-active and safe vaccine for protection of poultry from H5N1 highly pathogenic avian influenza viruses (HPAIVs) remains an important practical goal. In this study we used a low pathogenic wild aquatic bird virus isolate А/duck/Moscow/4182/2010 (H5N3) (dk/4182) as a live candidate vaccine. We compared this virus with four live 1:7 reassortant anti-H5N1 candidate vaccine viruses with modified hemagglutinin from either A/Vietnam/1203/04 (H5N1) or A/Kurgan/3/05 (H5N1) and the rest of the genes from either H2N2 cold-adapted master strain A/Leningrad/134/17/57 (rVN-Len and rKu-Len) or H6N2 virus A/gull/Moscow/3100/2006 (rVN-gull and rKu-gull). The viruses were tested in parallel for pathogenicity, immunogenicity and protective effectiveness in chickens using aerosol, intranasal and oral routes of immunization. All five viruses showed zero pathogenicity indexes in chickens. Viruses rVN-gull and rKu-gull were immunogenic and protective, but they were insufficiently attenuated and caused significant mortality of 1-day-old chickens. The viruses with cold-adapted backbones (rVN-Len and rKu-Len) were completely nonpathogenic, but they were significantly less immunogenic and provided lower protection against lethal challenge with HPAIV A/Chicken/Kurgan/3/05 (H5N1) as compared with three other vaccine candidates. Unlike other four viruses, dk/4182 was both safe and highly immunogenic in chickens of any age regardless of inoculation route. Single administration of 106 TCID50 of dk/4182 virus via drinking water provided complete protection of 30-days-old chickens from 100 LD50 of the challenge virus. Our results suggest that low pathogenic viruses of wild aquatic birds can be used as safe and effective live poultry vaccines against highly pathogenic avian viruses.

[Testing of apathogenic influenza virus H5N3 as a poultry live vaccine].

Four H5N2 experimental vaccine strains and the apathogenic wild duck H5N3 influenza virus A/duck/ Moscow/4182/2010 (dk/4182) were tested as a live poultry vaccine. Experimental strains had the hemagglutinin of the A/Vietnam/1203/04 strain lacking the polybasic HA cleavage site or the hemagglutinin from attenuated virus (Ku/ at) that was derived from the highly pathogenic influenza virus A/chicken/Kurgan/3/2005 (H5N1). The hemagglutinin of the Ku-at has the amino acid substitutions Asp54/Asn and Lys222/Thr in HA1 and Val48/Ile and Lys131/Thr in HA2, while maintaining the polybasic HA cleavage site at an invariable level. The other genes of these experimental strains were from the H2N2 cold-adapted master strain A/Leningrad/134/17/57 (VN-Len and Ku-Len) or from the apathogenic H6N2 virus A/gull/Moscow/3100/2006 (VN-Gull and Ku-Gull). A single immunization of mice with all tested strains elicited a high level of serum antibodies and provided complete protection against the challenge with the lethal dose of A/chicken/Kurgan/3/05. The pathogenicity indexes of the Ku-at and the other strains for chicken were virtually zero, whereas the index of the parent H5N1 virus A/chicken/Kurgan/3/2005 was 2.98. Intravenous, intranasal, and aerosol routes of vaccination were compared. It was shown that the strain dk/4182 was totally apathogenic for one-day-old chicken and provided complete protection against the highly pathogenic H5N1 virus.

What adaptive changes in hemagglutinin and neuraminidase are necessary for emergence of pandemic influenza virus from its avian precursor?

Wild ducks serve as the primary host for numerous and various influenza type A viruses. Occasionally, viruses from this reservoir can be transferred to other host species and cause outbreaks of influenza in fowl, swine, and horses, as well as result in novel human pandemics. Cellular tropism and range of susceptible host species are determined by interaction between virus and receptor molecules on cells. Here we discuss modern data regarding molecular features underlying interactions of influenza viruses with cellular receptors as well as a role for receptor specificity in interspecies transmission. By analyzing the earliest available pandemic influenza viruses (1918, 1957, 1968, 2009), we found that hemagglutinin reconfigured to recognize 2-6 sialic acid-containing receptors in the human upper airway tract together with altered enzymatic activity of neuraminidase necessary for maintaining functional balance with hemagglutinin are responsible for effective spread of influenza viruses in human populations. Resistance to low pH also contributes to this. Thus, a combination of such parameters makes it possible that influenza viruses give rise to novel pandemics.

Oligosaccharide specificity of influenza H1N1 virus neuraminidases.

A fluorescent neuraminidase (NA) assay has been developed; 20 samples in five replicates could be analyzed at the same time, allowing us to study the kinetics of the enzyme-substrate interaction. The specificities of six influenza H1N1 virus NAs for BODIPY-labeled 3'SiaLac, 3'SiaLacNAc, SiaLe(c), SiaLe(a), 6'SiaLac, and 6'SiaLacNAc were evaluated. The duck virus NA hydrolyzed 6'SiaLac and 6'SiaLacNAc 50 times more slowly than 2-3 isomers. Swine viruses digested SiaLe(a) and 2-6 sialosides 20 times more slowly than 2-3 trisaccharides. For the human viruses, the difference between 2-6 and 2-3 oligosaccharides desialylation efficiency did not exceed five times; notably, the inner core of 2-3 sialosaccharide was discriminated. The results are evidence that influenza virus NAs can distinguish substrate structure at the tri- and tetrasaccharide level.

Effect of gene constellation and postreassortment amino acid change on the phenotypic features of H5 influenza virus reassortants.

Reassortants between a low-pathogenic avian influenza virus strain A/Duck/Primorie/2621/2001 (H5N2) and a high-yield human influenza virus strain A/Puerto Rico/8/34 (H1N1) were generated, genotyped and analyzed with respect to their yield in embryonated chicken eggs, pathogenicity for mice, and immunogenicity. A reassortant having HA and NA genes from A/Duck/Primorie/2621/2001 virus and 6 genes from A/Puerto Rico/8/34 virus (6:2 reassortant) replicated efficiently in embryonated chicken eggs, the yields being intermediate between the yields of the avian parent virus and those of the A/Puerto Rico/8/34 parent strain. The reassortant having the HA gene from A/Duck/Primorie/2621/2001 virus and 7 genes from A/Puerto Rico/8/34 virus (7:1 reassortant) produced low yields. A variant of the 7:1 reassortant selected by serial passages in eggs had an amino acid substitution in the hemagglutinin (N244D, H3 numbering). The variant produced yields similar to those of the 6:2 reassortant. A 5:3 reassortant generated by a back-cross of the 6:2 reassortant with the avian parent and having PB1, HA and NA genes of A/Duck/Primorie/2621/2001 virus produced higher yields than the 7:1 or 6:2 reassortants, although still lower than the yields of A/Puerto Rico/8/34 virus. The 7:1, 6:2 and 5:3 reassortants were pathogenic for mice, with the level of virulence close to A/Puerto Rico/8/34 virus, in contrast to the extremely low pathogenicity of the A/Duck/Primorie/2621/2001 parent strain. Immunization of mice with an inactivated 6:2 H5N2 reassortant provided efficient immune protection against a reassortant virus containing the HA and NA genes of a recent H5N1 isolate. The results are discussed in connection with the problem of the improvement of vaccine strains against the threatening H5N1 pandemic.

Polymer-bound 6' sialyl-N-acetyllactosamine protects mice infected by influenza virus.

To develop a mouse model for testing receptor attachment inhibitors of human influenza viruses, the human clinical virus isolate in MDCK cells A/NIB/23/89M (H1N1) was adapted to mice by serial passaging through mouse lungs. The adaptation enhanced the viral pathogenicity for mice, but preserved the virus receptor binding phenotype, preferential binding to 2-6-linked sialic acid receptors and low affinity for 2-3-linked receptors. Sequencing of the HA gene of the mouse-adapted virus A/NIB/23/89-MA revealed a loss of the glycosylation sites in positions 94 and 163 of HA1 and substitutions 275Asp-->Gly in HA1 and 145Asn-->Asp in HA2. The four mouse strains tested differed significantly in their sensitivity to A/NIB/23/89-MA with the sensitivity increasing in the order of BALB/cJCitMoise, C57BL/6LacSto, CBA/CaLacSto and A/SnJCitMoise strains. Testing of protective efficacy of the polyacrylamide conjugate bearing Neu5Acalpha2-6Galbeta1-4GlcNAc trisaccharide under conditions of lethal or sublethal virus infection demonstrated a strong protective effect of this preparation. In particular, aerosol treatment of mice with the polymeric attachment inhibitor on 24-110 h after infection completely prevented mortality in sensitive animals and lessened disease symptoms in more resistant mouse strains.

H5N1 chicken influenza viruses display a high binding affinity for Neu5Acalpha2-3Galbeta1-4(6-HSO3)GlcNAc-containing receptors.

To characterize differences in the receptor-binding specificity of H5N1 chicken viruses and viruses of aquatic birds, we used a panel of synthetic polyacrylamide (PAA)-based sialylglycopolymers that carried identical terminal Neu5Acalpha2-3Gal fragments but varied by the structure of the next saccharide residues. A majority of duck viruses irrespective of their HA subtype, bound with the highest affinity to trisaccharide Neu5Acalpha2-3Galbeta1-3GlcNAc, suggesting that these viruses preferentially recognize sialyloligosaccharide receptors with type 1 core (Galbeta1-3GlcNAc). Substitution of 6-hydroxyl group of GlcNAc residue of tested sialylglycopolymers by 6-sulfo group had little effect on receptor binding by duck viruses. By contrast, H5N1 chicken and human viruses isolated in 1997 in Hong Kong preferred receptors with type 2 core (Galbeta1-4GlcNAcbeta) and bound sulfated trisaccharide Neu5Acalpha2-3Galbeta1-4(6-HSO3)GlcNAcbeta (6-Su-3'SLN) with the extraordinary high affinity. Another chicken virus, A/FPV/Rostok/34 (H7N1), and several mammalian viruses also displayed an increased affinity for sulfated sialyloligosaccharide receptor. The binding of chicken and mammalian viruses to tracheal epithelial cells of green monkey decreased after treatment of cells with glucosamine-6-sulfatase suggesting the presence of 6-O-Su-3'SLN determinants in the airway epithelium. It remains to be seen whether existence of the 6-O-Su-3'SLN groups in the human airway epithelial cells might facilitate infection of humans with H5N1 chicken viruses.

Receptor specificity of H5 influenza virus escape mutants.

The binding of viruses to synthetic polyacrylamide (PAA)-based sialylglycoconjugates was used to characterize the receptor specificities of antibody escape mutants of the influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2). The sialylglycoconjugates that were used carried identical terminal Neu5Acalpha2-3Gal moieties but differed in the structure of the next saccharide residue(s). Our data show that mutations in the vicinity of the haemagglutinin (HA) receptor-binding site (RBS) effect the recognition of the third saccharide residue and change the affinity pattern of binding. The affinity of the majority of the escape mutants for sialyl receptors increased compared to the parental strain.

Differences between influenza virus receptors on target cells of duck and chicken and receptor specificity of the 1997 H5N1 chicken and human influenza viruses from Hong Kong.

To study whether influenza virus receptors in chickens differ from those in other species, we compared the binding of lectins and influenza viruses with known receptor specificity to cell membranes and gangliosides from epithelial tissues of ducks, chickens, and African green monkeys. We found that chicken cells contained Neu5Ac alpha(2-6)Gal-terminated receptors recognized by Sambucus nigra lectin and by human viruses. This finding explains how some recent H9N2 viruses replicate in chickens despite their human virus-like receptor specificity. Duck virus bound to gangliosides with short sugar chains that were abundant in duck intestine. Human and chicken viruses did not bind to these gangliosides and bound more strongly than duck virus to gangliosides with long sugar chains that were found in chicken intestinal and monkey lung tissues. Chicken and duck viruses also differed by their ability to recognize the structure of the third sugar moiety in Sia2-3Gal-terminated receptors. Chicken viruses preferentially bound to Neu5Ac alpha(2-3)Gal beta(1-4)GlcNAc-containing synthetic sialylglycopolymer, whereas duck viruses displayed a higher affinity for Neu5Ac alpha(2-3)Gal beta(1-3)GalNAc-containing polymer. Our data indicate that sialyloligosaccharide receptors in different avian species are not identical and provide a potential explanation for the differences between the hemagglutinin and neuraminidase proteins of duck and chicken viruses.

Differences between HA receptor-binding sites of avian influenza viruses isolated from Laridae and Anatidae.

A comparative study of the hemagglutinin (HA) receptor binding site (RBS) of a number of H13 influenza viruses isolated from Laridae family of birds (gulls) and other influenza viruses obtained from the Anatidae family (ducks) was conducted. The affinity of all viruses to alpha N-acetylneuraminic acid (Neu5Ac alpha), 3'sialyllactose (3'SL), and sialylglycopolymers bearing 3'-sialyl(N-acetyllactosamine) (3'SLN-PAA), [Neu5Ac alpha(2-3)Gal beta(1-4)][-Fuc alpha(1-3)]GlcNAc beta (SLe(x)-PAA), and [Neu5Ac alpha(2-3)Gal beta(1-3)][-Fuc alpha(1-4)]GlcNAc beta (SLe(a)-PAA), was determined. The last three polymer glycoconjugates were synthesized for determining the contribution of carbohydrate chains after the galactose link to the binding with the receptor. The difference in affinity between 3'SL and Neu5Ac alpha in all studied H13 viruses is small, which indicates a less significant role of the galactose moiety in the binding to the receptor. The results of virus binding with polymer sialylglycoconjugates indicates that the method of linking, the third monosaccharide moiety, and the presence of an extra fucose substitute in this moiety may influence the binding considerably. For viruses isolated from ducks, the suitable polymer is SLe(a)-PAA (i.e., a 1-3 linkage between galactose and glucosamine is optimal). This finding is in accord with the data that H13 viruses isolated from the gulls differ based on their ability to interact with polymer sialylglycoconjugates. The affinity to all three polymers is uniform, and the presence of GlcNAc-linked fucose does not prevent the binding. A comparative analysis of six sequenced HA H13 viruses and other subtype viruses showed presence of substantial differences in the composition of amino acids of this region in H13 viruses.

Differences between influenza virus receptors on target cells of duck and chicken.

H5, H7, and H9 subtype influenza viruses in land-based poultry often differ from viruses of wild aquatic birds by deletions in the stalk of the neuraminidase, by the presence of additional carbohydrates on the hemagglutinin, and by occasional changes in the receptor specificity. To test whether these differences could reflect distinctions between the virus receptors in different avian species, we compared the binding of duck, chicken and human influenza viruses to cell membranes and gangliosides from epithelial tissues of duck, chicken and African green monkey. Human viruses bound to cell membranes of monkey and chicken but not to those of duck, suggesting that chicken cells unlike duck cells contain Sia(alpha2-6)Gal-terminated receptors recognized by human viruses. Duck virus bound to gangliosides with short sugar chains that were abundant in duck intestine. Human and chicken viruses did not bind to these gangliosides and bound more strongly than duck virus to gangliosides with long sugar chains that were found in chicken intestinal and monkey lung tissues. Our data suggest that the spectrum of sialylglycoconjugates which can serve as influenza virus receptors in chicken is more similar to the spectrum of receptors in the respiratory epithelia of monkey than to that in the epithelial tissues of duck. This notion could explain the recent emergence of avian H9N2 virus lineage with human virus-like receptor specificity and emphasizes the role of the chicken as a potential intermediate host for the transmission of viruses from aquatic birds to humans.