Вирусы гриппа птиц подтипов Н1 и Н5 участвовали в формировании высокопатогенных вариантов вирусов, вызвавших пандемии и панзоотии в XX–XXI веках. С целью оценки зоонозного потенциала вирусов этих подтипов, выделенных от диких уток в черте Москвы, была проведена адаптация вирусов к размножению в легких мышей, изучены их фенотипические свойства и идентифицированы генетические изменения, возникшие при адаптации. Изначально апатогенные для мышей вирусы A/duck/Moscow/4970/2013 (H1N1) и A/duck/Moscow/4182 C/2010 (H5N3) после 7–10 пассажей через легкие мышей изменили фенотип на патогенный. Полногеномное секвенирование выявило в адаптированных к мышам вирусах 2 аминокислотные замены в вирусе гриппа H1N1 (Glu627Lys в белке PB2 и Asp35Asn в гемагглютинине (HA) — нумерация по H3) и 6 мутаций в белках вируса H5N3 (Glu627Lys в PB2, Val113Ala в PB1, Ser82Pro в PB1 F2, Lys52Arg в HA2, Arg65Lys в NP и Ser59Ile в NA). Возрастание вирулентности для мышей, скорее всего, обусловлено общей для обоих вирусов заменой – Glu627Lys в белке PB2. Замена Asp35Asn в HA адаптированного к мышам вируса гриппа H1N1 ассоциирована с возрастанием значения рН конформационного перехода HA с 5.0 до 5.5 относительно HA дикого вируса. Обнаруженные в адаптированном варианте H5N3 мутации в белках НА, NA и PB1 F2 — уникальные. Мутации Glu627Lys в PB2, Arg65Lys в NP и Val113Ala в PB1, скорее всего, носят адаптационный характер.

Avian influenza viruses of the H1 and H5 subtypes were involved in the formation of highly pathogenic viruses that caused pandemics and panzootics in the 20th–21st centuries. In order to assess the zoonotic potential of viruses of these subtypes, two viruses of the H1N1 and H5N3 subtypes have been isolated from wild ducks in Moscow and adapted for growth in mouse lungs. Their phenotypic properties were studied, and the genetic changes that occurred during adaptation were identified. The original A/duck/Moscow/4970/2013 (H1N1) and A/duck/Moscow/4182-C/2010 (H5N3) viruses were apathogenic for mice but became pathogenic after 7–10 passages in mouse lungs. Complete genome sequencing revealed 2 amino acid substitutions in the proteins of the H1N1 mouse-adapted variant (Glu627Lys in PB2 and Asp35Asn in hemagglutinin (HA) – numbering according to H3) and 6 mutations in the proteins of H5N3 virus (Glu627lys in PB2, Val113Ala in PB1, Ser82Pro in PB1-F2, Lys52Arg in HA2, Arg65Lys in NP, and Ser-59Ile in NA). The increase in virulence is most likely due to a Glu627Lys substitution in the protein PB2 found in both viruses. The replacement Asp35Asn in HA of the mouse-adapted H1N1 virus is associated with an increase in the pH value of the HA transition to 5.5 versus 5.0 for that of the wild virus. The mutations found in the HA, NA, and PB1-F2 proteins of the adapted H5N3 variant are unique. The mutations Glu627Lys in PB2, Arg65Lys in NP, and Val113Ala in PB1 are most likely host adaptive.

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.

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.

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.

ABSTRACT Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin H5 and H7 subtypes emerge after introduction of low-pathogenic avian influenza viruses (LPAIVs) from wild birds into poultry flocks, followed by subsequent circulation and evolution. The acquisition of multiple basic amino acids at the endoproteolytical cleavage site of the hemagglutinin (HA) is a molecular indicator for high pathogenicity, at least for infections of gallinaceous poultry. Apart from the well-studied significance of the multibasic HA cleavage site, there is only limited knowledge on other alterations in the HA and neuraminidase (NA) molecules associated with changes in tropism during the emergence of HPAIVs from LPAIVs. We hypothesized that changes in tropism may require alterations of the sialyloligosaccharide specificities of HA and NA. To test this hypothesis, we compared a number of LPAIVs and HPAIVs for their HA-mediated binding and NA-mediated desialylation of a set of synthetic receptor analogs, namely, α2-3-sialylated oligosaccharides. NA substrate specificity correlated with structural groups of NAs and did not correlate with pathogenic potential of the virus. In contrast, all HPAIVs differed from LPAIVs by a higher HA receptor-binding affinity toward the trisaccharides Neu5Acα2-3Galβ1-4GlcNAcβ (3′SLN) and Neu5Acα2-3Galβ1-3GlcNAcβ (SiaLec) and by the ability to discriminate between the nonfucosylated and fucosylated sialyloligosaccharides 3′SLN and Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ (SiaLex), respectively. These results suggest that alteration of the receptor-binding specificity accompanies emergence of the HPAIVs from their low-pathogenic precursors. IMPORTANCE Here, we have found for the first time correlations of receptor-binding properties of the HA with a highly pathogenic phenotype of poultry viruses. Our study suggests that enhanced receptor-binding affinity of HPAIVs for a typical “poultry-like” receptor, 3′SLN, is provided by substitutions in the receptor-binding site of HA which appeared in HA of LPAIVs in the course of transmission of LPAIVs from wild waterfowl into poultry flocks, with subsequent adaptation in poultry. The identification of LPAIVs with receptor characteristics of HPAIVs argues that the sialic acid-binding specificity of the HA may be used as a novel phenotypic marker of HPAIVs.

The influenza virus hemagglutinin (HA) is an envelope virus glycoprotein responsible for the attachment of the virus particles to cells via binding terminal sialic acid residues of cell surface oligosaccharides. In our previous works on influenza A virus escape mutants, that is, mutants resistant to the neutralization effect of monoclonal antibodies, we encountered amino acid changes in the vicinity of receptor-binding pocket of the HA. In this work the degree of the affinity to both alpha-2, -3, and alpha-2, -6, -sialoglycoconjugates was assessed for escape mutants of influenza H1 and H5 viruses. The data demonstrate that the decrease of the positive electrostatic charge of the HA molecule surface resulting from amino acid changes conferring resistance to monoclonal antibodies may lead to a lowering of the affinity to sialic acid-containing analogs of cell receptors. The results are discussed in the context of the evolution of HA in natural circulation of H1 and H5 influenza viruses.

The influenza virus hemagglutinin (HA) is an envelope virus glycoprotein responsible for the attachment of the virus particles to cells via binding terminal sialic acid residues of cell surface oligosaccharides. In our previous works on influenza A virus escape mutants, that is, mutants resistant to the neutralization effect of monoclonal antibodies, we encountered amino acid changes in the vicinity of receptor-binding pocket of the HA. In this work the degree of the affinity to both alpha-2, -3, and alpha-2, -6, -sialoglycoconjugates was assessed for escape mutants of influenza H1 and H5 viruses. The data demonstrate that the decrease of the positive electrostatic charge of the HA molecule surface resulting from amino acid changes conferring resistance to monoclonal antibodies may lead to a lowering of the affinity to sialic acid-containing analogs of cell receptors. The results are discussed in the context of the evolution of HA in natural circulation of H1 and H5 influenza viruses.

24ВведениеГликопротеины оболочки изменениямивируса гриппа, гемагглю-тинин (НА) и нейраминидаза (NA), определяющие иммунный ответ, обладают значительной вариабель-ностью. В человеческой популяции Нв настоящее вре-мя циркулируют вирусы гриппа А, НА эскейпкоторых отно-сятся к двум подтипам: Н1 и Н3. Новый вариант ви-руса гриппа А подтипа H1N1, возникший в результате Секвенированиескрещивания двух вирусов гриппа свиней, североаме-риканского резистентностии европейского [9, 21], вызвал пандемию в 2009 г. Другие подтипы НА (Н2, Н4—Н16) сейчас не НАпредставлены в человеческой манапопуляции, они цирку-лируют среди диких и домашних птиц куи млекопитаю-щих. Однако вирус подтипа H5N1 с 2003 г. вызывает вирусовэпизоотии с высокой смертностью рецепторсвязывающегоу птиц и споради-ческие случаи тяжелого заболевания людей. В связи шихс этим заноисследование вариаций антигенной специфич-ности НА подтипов Н1 и сродстН5, а также вариаций тех

A panel of 6 neutralizing monoclonal antibodies (MAbs) raised against A/Moscow/IIV01/2009 (H1N1) virus isolated during the 2009 pandemic was used for the selection of 26 escape mutants. The mutants were characterized in immune cross-reactions with the panel of MAbs. The sequencing of the mutant HA genes revealed 5 amino acid positions recognized by monoclonal antibodies: 129, 156, 158, 159, and 190 (H3 numbering). The amino acid positions were distributed in two epitopes belonging to antigenic sites Sa and Sb. The mutant HAs exhibited variations in the affinity to synthetic high molecular mass sialic acid-containing receptor analogues. Results are discussed in connection with the antigenic drift potential of the "swine-like" pandemic 2009 influenza virus.

Background The viral glycoproteins, HA and NA, expressed on the surface of influenza virions play an important role in determining pathogenic properties of this virus. Influenza virus infection is initiated by specific interactions between the viral HA and terminal sialic acid-containing molecules of cell surface receptors mediating the virus absorption on the target cells. The NA cleaves off the terminal sialic acid residues from the host cell promoting the release of virus progeny and preventing the formation of virion aggregates at the budding site. The functional balance between HA and NA is prerequisite of successful influenza virus replication. Human infections caused by highly pathogenic avian influenza viruses emphasize a need for the assessment of factors that allow adaptation of avian viruses (AIV) to humans. Here, in order to clear a possible role of HA and NA as well as their balanced action in pathogenic properties of AIV, we evaluated HA receptor binding and NA substrate specificities of high and low pathogenic avian influenza (HPAI and LPAI) viruses of H5N1, H5N9, H5N2, H7N7 and H9N2 subtypes for a set of synthetic sialooligosaccharides which are analogues of the natural influenza virus receptors. Methods Receptor binding specificity of HA was investigated in a direct binding assay as described previously (Matrosovich et al., 2000). In brief, 96-well plates were coated with purified virus for 16 h at 4°C. After that biotinylated sialyloligosaccharides in TN buffer containing neuraminidase inhibitor were added. Following the addition of Strept-POD and ABTS substrate solution, optical density at 405 nm was determined. The affinity constants (Kaff) were determined as sialic acid concentration (μM-1) at the point Amax/2 on Scatchard plots. The fluorescent assay for studying the substrate specificity of neuraminidase was described earlier (Mochalova et al., 2005). The method based on the a quantitative separation of neutral fluorescent-labeled product from negatively charged fluorescent-labeled uncleaved substrate using anion exchanger microcartridges. Fluorescence was measured at 485/535 nm. Virus NA specificity for each sialoside was calculated as the slope of the starting linear region of the Vo versus So curve (Vo - initial rate of the desialylation, So - initial substrate concentration). Results HA receptor binding and NA substrate oligosaccharide specificity are usually defined as ability of the viral HA or NA to distinguish the type of bond between terminal neuraminic acid and galactose residues. HPAI viruses displayed a higher (2-12 times) HA affinity and higher (2-8 times) NA hydrolytic efficiency than LPAI viruses towards trisaccharides Neu5Acα2-3Gals1-4GlcNAcs (3’SLN) and Neu5Acα2-3Gals1-3GlcNAcs (SiaLec). Furthermore, HAs of HPAI and LPAI viruses differed in their ability to discriminate non-fucosylated and fucosylated glycans, namely, 3’SLN vs. Neu5Acα2-3Gals1-4(Fucα1-3)GlcNAcs?(SiaLex). When receptor binding specificity was plotted as 3’SLN/SiaLex ratio, the values for all HPAI viruses ranged between 3 - 5 whereas for LPAI viruses these values were only about 2 or lower. We hypothesize that the optimal ratio of affinities towards 3’SLN and SiaLex is an important modulator of pathogenicity. Conclusion In summary, this study presents combined data on HA and NA specificity of HPAI and LPAI viruses. A combination of both methods allows for a rapid monitoring of changes in the OS specificity of both HA and NA. Correlation of these data with mutational analyses of the glycoprotein genes promise to be a powerful tool for the prediction of new pandemic strains.

Influenza virus neuraminidase inhibitors (NAIs), currently used as anti-influenza drugs, can lead to the appearance of drug-resistant variants. Resistance to NAIs appears due to mutations in the active site of the neuraminidase (NA) molecule that decrease the NA enzymatic activity and sometimes in the hemagglutinin (HA) that decrease its affinity for cell receptors and, therefore, reduce the requirement for NA activity in releasing mature virions from infected cells. Using a set of sialo-oligosaccharides, we evaluated changes in the receptor-binding specificity of the HA and substrate specificity of the NA of influenza B viruses that had acquired resistance to NAIs. The oligosaccharide specificity of two pairs of field influenza B viruses, namely: i) B/Memphis/20/96 and its NAI-resistant variant, B/Memphis/20-152K/96, containing mutation R152K in the NA and 5 amino acid substitutions in the HA1, and ii) B/Hong Kong/45/2005 and its NAI-resistant variant B/Hong Kong/36/2005, containing a single R371K mutation in the NA, was evaluated. Wild type viruses bound strictly to a “human type” receptor, α2-6-sialo-oligosaccharide 6`SLN, but desialylated it is approximately 8 times less efficiently than the α2-3 sialosaccharides. Both drug-resistant viruses demonstrated the ability to bind to “avian type” receptors, α2-3 sialo-oligosaccharides (such as 3`SLN), whereas their affinity for 6`SLN was noticeably reduced in comparison with corresponding wild type viruses. Thus, the development of the NAI resistance in the studied influenza B viruses was accompanied by a readjustment of HA-NA oligosaccharide specificities.

The structure of the influenza virus neuraminidases, the spatial organization of their active site, the mechanism of carbohydrate chains desialylation by neuraminidase, and its role in the influenza virus function at different stages of the viral infectious cycle are considered in this review. Data on the neuraminidase substrate specificity and different approaches in studying the activity of this enzyme are summarized. In addition, data on neuraminidase inhibitors (as antivirals) are provided, along with considerations on the mechanisms of resistance of modern influenza viruses to those antivirals.

It has recently been shown that the influenza virus can specifically bind the residue of a nonsialylated sulfated oligosaccharide Gal(6SO3H)β1-4GlcNAcβ (6’SLacNAc). To identify by photoaffinity labeling the virion component that binds 6’SLacNAc, we synthesized a carbohydrate probe containing a 125I labeled diazocyclopentadien-2-yl carbonyl group as an aglycone. According to the electrophoretic data, the labeled areas corresponded to a large hemagglutinin subunit, a nucleocapsid protein, and neuraminidase (NA). Probing in the presence of an excess of 6’SLacNAcβ-OCH2CH2NHAc glycoside resulted in redistribution of the labeling intensity, with the maximum inhibition being observed for NA. The data obtained indicate that NA is a viral 6’SLacNAc-binding protein.

Balanced action of hemagglutinin (HA) and neuraminidase (NA) is an important condition of influenza virus efficient replication, but a role of HA and NA specificities at oligosaccharide level in maintaining such a balance remains poorly studied. Avian virus HA binds exclusively and NA digests efficiently α2–3-sialylated carbohydrate chains, while human virus HA interacts with α2–6 chains and low-active NA cleaves both α2–3- and α2–6-sialosides. Reassortment between viruses leading to appearance of avian virus HA and human virus NA on the virion surface often resulted in decreasing the replicative potential of the formed variants because of disturbance of a functional balance between “alien” HA and NA. A restoration of the reassortant productivity happened due to the appearance of amino acid substitutions in HA and, sometimes, NA. Here, a role of NA and HA oligosaccharide specificities in a restoration of HA–NA functional balance in high-yield passage variants was studied. Postreassortment changes in HA receptor-binding and NA substrate specificities for three reassortant/passage variant virus pairs towards 3′SiaLac, 3′SiaLacNAc, SiaLec, SiaLea, SiaLex, 6′SiaLac, and 6′SiaLacNAc were determined. Selection of the high-yield variants of the human-avian reassortants led either to twofold decrease in the affinity of HA for most α2–3-sialosides and the appearance of affinity for α2–6-sialosides (H3N2 reassortant), or to decreasing the HA affinity for SiaLec and SiaLea (H3N1 reassortant), or to enhancing the ability of NA to discriminate between α2–3/2–6 substrates (H4N1 reassortant). Thus, all postreassortment changes in oligosaccharide specificities of “alien” HA and NA were directed towards their adjustment to each other, but by different manner.

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.