[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.

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.

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.

[Different receptor specificity of influence viruses from ducks and chickens and its reflection in the composition of sialosides on host cells and mucins].

The ability of influenza viruses from different hosts to bind to the intestinal epithelium of various birds (Anseriformes (Anatidae), Galliformes, Charadriiformes (sandpipers and sea gulls), Ciconiiformes (storks), Podicipediformes (grebes), and Gruiformes was studied. The composition of sialo-containing receptors on the epithelia was examined, by using lectins. Intestinal epitheliocytes of the Anatidae (Anseriformes) family was shown to have a low content of receptors binding both Sambucus nigra agglutinin (SNA) lectin specific to Siaalpha-6Gal, and Maackia amurensis agglutinin (MAA) lection specific to Siaalpha2-2Gal. Nevertheless, these cells well bound duck influenza viruses. The intestinal epithelium of Ciconiiformes, Podicipediformes, and Gruiformes well bound MMA lection, but avian influenza viruses weakly bound the latter. The intestinal cells of Gallinaceae bound both MMA and SNA lectins and avian and human influenza viruses. Thus, the composition of natural sialosides is different in various avian species whereas the receptor specificity of influenza viruses from various hosts reflects these differences. This can be accounted for by the differences in the ability of influenza viruses from different birds to break through the interspecies barrier, infecting mammals and human beings in particular.

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.

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.

[The effect of losing glycosylation sites near the receptor-binding region on the receptor phenotype of the human influenza virus H1N1]. / Vliianie utraty saitov glikozilirovaniia, raspolozhennykh vblizi retseptor-sviazyvaiushchego uchastka, na retseptornyi fenotip H1N1 virusa grippa cheloveka.

The receptor properties of influenza virus (IF) isolates/SSSR/90/77 are studied. The isolates are peculiar for losing glycosylation sites (GS) at the Asn131 receptor-binding region (GS131) after passaging in mice and at the Asn158 region (GS158) after cultivation in the presence of mouse serum. The loss of each carbohydrate residue increases the influenza virus affinity for carbohydrate chains with the terminal group Neu5Ac alpha 2-6Gal and reduces its affinity for Neu5Ac alpha 2-3Gal receptors. The effect is more pronounced in the GS158-depleted virus. Upon substitution of asparagine by aspartic acid, the electrostatic component of virus binding to the receptor is altered because of the increased negative charge on hemagglutinin. The virus receptor phenotype changes depending on the cultivation conditions. The isolate adapted to mice has higher affinity to mouse lung cell receptors, while the virus propagated in chick embryos in the presence of inhibitors has higher affinity to allantoic membrane cells.

[Differences in receptor specificity between the influenza A viruses isolated from the duck, chicken, and human]. / Sravnenie retseptornoi spetsifichnosti virusov grippa A, vydelennykh ot utok, kur i cheloveka.

The affinity of the duck, chicken, and human influenza viruses to the host cell sialosides was determined, and considerable distinctions between duck and chicken viruses were found. Duck viruses bind to a wide range of sialosides, including the short-stem gangliosides. Most of the chicken viruses, like human ones, lose the ability to bind these gangliosides, which strictly correlates with the appearance of carbohydrate at position 158-160. The affinity of the chicken viruses to sialoglycoconjugates of chicken intestine as well as chicken, monkey, and human respiratory epithelial cells exceeds that of the duck viruses. The human influenza viruses have high affinity to the same cells but do not bind at all to the duck epithelial cell. This testifies to the absence of 6'-sialylgalactose residues from the duck cells, in contrast to chicken and monkey cells. The alteration of the receptor specificity of chicken viruses in comparison with duck ones results in the similarity of the patterns of accessible cells for chicken and human influenza viruses. This may be the cause of the appearance of the line of H9N2 viruses from Hong Kong live bird markets with receptor specificity similar to that of H3N2 human viruses, and of the ability of H5N1 and H9N2 chicken influenza viruses to infect humans.

Polymeric inhibitor of influenza virus attachment protects mice from experimental influenza infection.

Synthetic sialic acid-containing macromolecules inhibit influenza virus attachment to target cells and suppress the virus-mediated hemagglutination and neutralize virus infectivity in cell culture. To test the protective effects of attachment inhibitors in vivo, mice were infected with mouse-adapted influenza virus A/Aichi/2/68 (H3N2) and treated with synthetic polyacrylamide-based sialylglycopolymer PAA-YDS bearing moieties of (Neu5Acalpha2-6Galbeta1-4GlcNAcbeta1-2Manalpha1)2-3,6Manbeta1-4GlcNAcbeta1-4GlcNAc. Single intranasal inoculations with PAA-YDS 30 min before or 10 min after infection increased the survival of mice (P<0.01). Multiple treatments with aerosolized PAA-YDS on days 2-5 post infection also increased survival (P<0.01), alleviated disease symptoms, and decreased lesions in the mouse lungs. These data suggest that synthetic polyvalent inhibitors of virus attachment can be used for prevention and treatment of influenza.

H9N2 influenza A viruses from poultry in Asia have human virus-like receptor specificity.

H9N2 influenza A viruses are currently widespread in chickens, quail, and other poultry in Asia and have caused a few cases of influenza in humans. In this study, we found that H9N2 viruses from Hong Kong live bird markets have receptor specificity similar to that of human H3N2 viruses. In addition, the neuraminidase of poultry H9N2 viruses has mutations in its hemadsorbing site, a characteristic resembling that of human H2N2 and H3N2 viruses but differing from that of other avian viruses. Peculiar features of surface glycoproteins of H9N2 viruses from Hong Kong suggest an enhanced propensity for introduction into humans and emphasize the importance of poultry in the zoonotic transmission of influenza viruses.

Selection of influenza virus mutants in experimentally infected volunteers treated with oseltamivir.

Volunteers experimentally infected with influenza A/Texas/36/91 (H1N1) virus and treated with the neuraminidase (NA) inhibitor oseltamivir were monitored for the emergence of drug-resistant variants. Two (4%) of 54 resistant viruses were detected by NA inhibition assay among last-day isolates recovered from 54 drug recipients. They bore a substitution His274Tyr in the NA. Hemagglutinin (HA) variants detected in the placebo group differed from the egg-adapted inoculum virus by virtue of amino acid substitutions at residues 137, 225, or both. These variants had a higher affinity for Neu5Ac(alpha2-6)Gal-containing receptors, which are characteristic of human respiratory epithelium, than for Neu5Ac(alpha2-3)Gal-containing receptors, which are typical of chicken egg allantoic membrane. Although appearing to be more sensitive to oseltamivir in humans, the variants with increased affinity for Neu5Ac(alpha2-6)Gal receptors were less sensitive than the Neu5Ac(alpha2-3)Gal-binding variants in Madin-Darby canine kidney cells. Thus, HA affinity for receptors is an essential feature of influenza virus susceptibility to NA inhibitors, both in cell culture and in humans.

Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus.

The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.

[The evaluation of the reactogenicity, harmlessness and prophylactic efficacy of Grippol trivalent polymer-subunit influenza vaccine administered to schoolchildren]. / Otsenka reaktogennosti, bezvrednosti i profilakticheskoi éffektivnosti grippoznoi trivalentnoi polimer-sub''edinichnoi vaktsiny "Grippol" pri vvedenii detiam shkol'nogo vozrasta.

Vaccine "Grippol"--has been developed at the State Research Center--Institute of Immunology. The preparation belongs to new generation vaccines and is a trivalent polymer-subunit vaccine containing the sterile conjugate of influenza virus surface proteins, types A and B, bound with copolymer polyoxidonium. The administration of "Grippol" to children of school age (6-18 years) demonstrated low reactogenicity of the vaccine, its safety and sufficient prophylactic effectiveness. During observations on total morbidity (with the exception of influenza and acute respiratory diseases) no side effects produced by "Grippol" were registered. At the same time the fact that the morbidity rate of upper respiratory tract disease in the group of children immunized with the vaccine decreased in comparison with the control group (by 2.4 times) cannot be disregarded.

Intergenic HA-NA interactions in influenza A virus: postreassortment substitutions of charged amino acid in the hemagglutinin of different subtypes.

In our previous studies influenza A virus reassortants having neuraminidase (NA) gene of A/USSR/90/77 (H1N1) strain and hemagglutinin (HA) genes of H3, H4 and H13 subtypes were shown to produce a low virus yield and to exhibit a strong tendency to virion aggregation. More detailed studies with the use of a H3N1 reassortant and its high-yield non-aggregating variants revealed that NA of A/USSR/90/77 strain is inefficient in the removal of the terminal sialic acid residues from the virion components, and that the inefficiency of NA may be compensated by mutations in HA gene leading to a decrease of the receptor-binding affinity (Kaverin, N.V. , Gambaryan, A.S., Bovin, N.V., Rudneva, I.A., Shilov, A.A., Khodova, O.M., Varich, N.L., Sinitsin, B.V., Makarova, N.L., Kaverin, N.V., 1998. Postreassortment changes in influenza virus hemagglutinin restoring HA-NA functional match, Virology 244, 315-321). The present report describes studies performed with the use of H2N1 and H4N1 reassortants having HA genes of A/Pintail/Primorie/695/76 (H2N3) and A/Duck/Czechoslovakia/56 (H4N6) strains respectively and NA gene of A/USSR/90/77 strain. The low-yield reassortants and their high-yield non-aggregating variants were studied in both direct and competitive binding assays with sialic acid-containing substrates. The non-aggregating variants were shown to have a decreased affinity as compared to the initial reassortants toward high-molecular-weight sialic acid-containing substrates. The sequencing of HA genes revealed that all non-aggregating variants of H2N1 and H4N1 reassortants had amino acid substitutions increasing the negative charge of the HA molecule in the vicinity of the receptor-binding pocket. The results suggest that the influenza virus reassortants containing low-functional NA undergo similar postreassortment changes irrespective of the HA subtype: their receptor-binding activity decreased due to negatively charged amino acid substitutions in the vicinity of the receptor-binding pocket.

Selection of receptor-binding variants of human influenza A and B viruses in baby hamster kidney cells.

Cultivation of human influenza viruses in the allantoic cavity of embryonated chicken eggs leads to a selection of receptor-binding variants with amino acid substitutions on the globular head of the hemagglutinin (HA) molecule. Such selection can be avoided by growing the human viruses in Madin Darby canine kidney (MDCK) cells. In the present study, we tested whether baby hamster kidney (BHK) cells select receptor-binding mutants of human influenza viruses. After isolating H1N1, H3N2, and type B influenza viruses from clinical samples in MDCK cells, we passaged them in either BHK cells or chicken eggs. The BHK-grown viruses differed from their MDCK-grown counterparts by virtue of mutations in the HA: 225D --> G (H1N1 virus), 128T --> A and 226I --> V (H3N2), and 187N --> D (type B) (H3 numbering). Variants with different substitutions were selected by passaging of the same MDCK-grown parents in eggs: 141L --> H, 208R --> H, and 225D --> G (H1N1), 194L --> I (H3N2), and 137G --> R (B). Compared with their MDCK-grown counterparts, both BHK- and egg-grown viruses possessed a higher affinity for the cellular membranes of BHK cells and of the chorioallantoic cells of chicken embryos and for a 3'-sialylgalactose-containing synthetic sialylglycopolymer. By contrast, changes in the affinity of mutants for a 6'-sialyl-(N-acetyllactosamine)-containing sialylglycopolymer varied from negative to positive. Fluorescence-activated cell-sorting analysis with linkage-specific lectins showed that the density of the 6'-sialyl-(N-acetyllactosamine)-containing receptors is substantially lower on the surface of BHK cells than on MDCK cells, providing an explanation for the growth restriction of human viruses in the former cells. Our data demonstrate that cultures of BHK cells, like eggs, can select receptor-binding variants of human influenza viruses.

Effects of egg-adaptation on the receptor-binding properties of human influenza A and B viruses.

Propagation of human influenza viruses in embryonated chicken eggs (CE) results in the selection of variants with amino acid substitutions near the receptor-binding site of the hemagglutinin (HA) molecule. To evaluate the mechanisms by which these substitutions enable human virus growth in CE, we studied the binding of 10 human influenza A (H1N1, H3N2) and B strains, isolated and propagated solely in MDCK cells, and of their egg-adapted counterparts to preparations of cellular membranes, gangliosides, sialylglycoproteins, and sialyloligosaccharides. All egg-adapted variants differed from nonadapted strains by increased binding to the plasma membranes of chorio-allantoic (CAM) cells of CE and by the ability to bind to CAM gangliosides. In addition, there was no decrease in affinity for inhibitors within allantoic fluid. These findings indicate that growth of human influenza viruses in CE is restricted because of their inefficient binding to receptors on CAM cells and that gangliosides can play an important role in virus binding and/or penetration. The effects of the egg-adaptation substitutions on the receptor-binding properties of the viruses include (i) enhancement of virus binding to the terminal Sia(alpha2-3)Gal determinant (substitutions in HA positions 190, 225 of H1N1 strains and in position 186 of H3N2 strains); (ii) a decrease of steric interference with more distant parts of the Sia(alpha2-3Gal)-containing receptors (a loss of glycosylation sites in positions 163 of H1 HA and 187 of type B HA); and (iii) enhanced ionic interactions with the negatively charged molecules due to charged substitutions at the tip of the HA [187, 189, 190 (H1), and 145, 156 (H3)]. Concomitantly with enhanced binding to Sia(alpha2-3)Gal-terminated receptors, all egg-adapted variants decreased their affinity for equine macroglobulin, a glycoprotein bearing terminal 6'-sialyl(N-acetyllactosamine)-moieties.

Evidence for zanamivir resistance in an immunocompromised child infected with influenza B virus.

Zanamivir, a neuraminidase inhibitor, has shown promise as a drug to control influenza. During prolonged treatment with zanamivir, a mutant virus was isolated from an immunocompromised child infected with influenza B virus. A hemagglutinin mutation (198 Thr-->Ile) reduced the virus affinity for receptors found on susceptible human cells. A mutation in the neuraminidase active site (152 Arg-->Lys) led to a 1000-fold reduction in the enzyme sensitivity to zanamivir. When tested in ferrets, the mutant virus had less virulence than the parent; however, it had a growth preference over the parent in zanamivir-treated animals. Despite these changes, the sensitivity of the mutant virus to zanamivir assessed by a standard test in MDCK cells was unaffected. These data indicate that the current methods for monitoring resistant mutants are potentially flawed because no tissue culture system adequately reflects the receptor specificity of human respiratory tract epithelium.

Effects of host-dependent glycosylation of hemagglutinin on receptor-binding properties on H1N1 human influenza A virus grown in MDCK cells and in embryonated eggs.

There is growing evidence that the receptor-binding characteristics of influenza viruses are affected by the host-dependent glycosylation of viral hemagglutinin (HA). To better understand these effects, we propagated two variants of the human influenza virus USSR/90/77 (which differed by the mutation Asn131 reversible Asp131 in the glycosylation sequon of their HA) in either embryonated chicken eggs or MDCK cell. Those variants were then compared for their ability to bind soluble receptor analogs and to attach to receptors represented on a solid phase. The carbohydrate chain at position 131 of the HA (CHO 131) interfered with virus binding to soluble Sia2-6Gal-containing macromolecular receptors, but had little or no effect on its binding to Sia2-3Gal-containing macromolecules. This specificity could be explained by the different orientation of the asialic parts of the 2-3-linked sialosides versus 2-6-linked sialosides with respect to the receptor-binding site (Eisen et al., 1997, Virology 232, 19-31). In the case of virus attachment to solid-phase immobilized receptors, MDCK-grown viruses bound substantially more weakly than their egg-grown counterparts to receptors of avian origin, whereas binding to mammalian cell membranes was only marginally affected by differences in host-specific glycosylation of the virus. Our data indicated that the effects of the carbohydrate side chain of HA on virus receptor-binding activity are dependent on both the cells in which the virus was grown and the nature of the cellular receptors or intercellular inhibitors to which the virus binds.