Pleiotropic effects of hemagglutinin amino acid substitutions of influenza A(H1N1)pdm09 virus escape mutants.

In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We investigated pH optimum of fusion, temperatures of HA heat inactivation, in vivo and in vitro replication kinetics, and connectivity with panel of sera of survivors patients in different epidemic seasons of the previously obtained influenza H1 escape mutants. Our results showed that N133D (H3 numbering) mutation significantly lowered the pH of fusion optimum.

Predicting the Evolutionary Variability of the Influenza A Virus.

The influenza A virus remains one of the most common and dangerous human health concerns due to its rapid evolutionary dynamics. Since the evolutionary changes of influenza A viruses can be traced in real time, the last decade has seen a surge in research on influenza A viruses due to an increase in experimental data (selection of escape mutants followed by examination of their phenotypic characteristics and generation of viruses with desired mutations using reverse genetics). Moreover, the advances in our understanding are also attributable to the development of new computational methods based on a phylogenetic analysis of influenza virus strains and mathematical (integro-differential equations, statistical methods, probability-theory-based methods) and simulation modeling.

Pleiotropic effects of amino acid substitutions in H5 hemagglutinin of influenza A escape mutants.

We believe that the monitoring of pleiotropic effects of the hemagglutinin (HA) mutations found in H5 escape mutants is essential for accurate prediction of mutants with pandemic potential. In the present study, we assessed multiple characteristics of antibody-selected HA mutations. We examined the pH optimum of fusion, HA heat inactivation, affinity to sialyl receptors, and in vitro and in vivo replication kinetics of various influenza H5 escape mutants.

[Effect of amino acid substitutions in small subunit of avian H5N2 influenza virus hemagglutinin on selection of the mutants resistant to neutralizing monoclonal antibodies].

Changes associated with the resistance to physical and chemical factors in the hemagglutinin (HA) of influenza A viruses may play an important role in the selection of different influenza variants during circulation in nature. Here, we studied the escape mutants of influenza virus A/mallard/Pennsylvania/10218/84 (H5N2) that were selected by the monoclonal antibody. The escape mutant m4F11(4) carried a single amino acid substitution in large subunit (HA1) of the HA, S145P1, and two ones, m4G10(10) and m4G10(6), had additional amino acid changes in the small subunit (HA2), namely: L124F2 and L124F2 + N79D2, respectively.

Location and architecture of an antibody-binding site of influenza A virus nucleoprotein.

Amino acid positions recognized by monoclonal antibodies (MAbs) in the influenza A virus nucleoprotein (NP) have been reported. As these residues were scattered in the three-dimensional (3D) structure of NP, no patterns of the architecture of antibody-binding sites could be inferred. Here, we used site-specific mutagenesis and ELISA to screen the amino acids surrounding position 470 recognized by the MAb 3/1 as a linear epitope.

Pleiotropic effects of hemagglutinin amino acid substitutions of H5 influenza escape mutants.

In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We examined pH optimum of fusion, temperatures of HA heat inactivation, and in vitro and in vivo replication kinetics of the previously obtained influenza H5 escape mutants. Our results showed that HA1 N142K mutation significantly lowered the pH of fusion optimum.

[Effect of mutations changing the antigenic specificity on the receptor-binding activity of the influenza virus hemagglutinin of H1 and H5 subtypes].

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.

Yields of virus reassortants containing the HA gene of pandemic influenza 2009 virus.

influenza virus; reassortment; virus yield; gene constellation.

Escape mutants of pandemic influenza A/H1N1 2009 virus: variations in antigenic specificity and receptor affinity of the hemagglutinin.

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).

[High-yield reassortant virus containing hemagglutinin and neuraminidase genes of pandemic influenza A/Moscowl/01/2009 (H1N1) virus].

The crossing of influenza A/Moscow/01/2009 (H1N1) virus and reassortant strain X31 (H3N2) containing the genes of internal and non-structural proteins of A/Puerto Rico/8/34 (H1N1) strain gave rise to reassortant virus ReM8. The reassortant contained hemagglutinin (HA) and neuraminidase (NA) genes of pandemic 2009 influenza virus and 6 genes of high-yield A/Puerto Rico/8/34 (H1N1) strain. The reassortant ReM8 produced higher yields in the embryonated chicken eggs than the parent pandemic virus, as suggested by infectivity and HA activity titration as well as by ELISA and the measurement of HA protein content by scanning electrophoresis in polyacrylamide gel slabs.

Antibody-binding epitope differences in the nucleoprotein of avian and mammalian influenza A viruses.

Abstract Influenza virus nucleoprotein (NP) binds to the viral genome RNA and forms the internal ribonucleoprotein complex of the virus particle. Avian and human influenza virus NP have characteristic differences at several amino acid positions. It is not known whether any of these differences can be recognized by antibodies.

[Impact of mutations in the hemagglutinin gene of H5N1 influenza virus on antigenicity and virulence as revealed by site-specific mutagenesis].

In our earlier studies, we have shown that amino acid changes in the hemagglutinin (HA) of influenza H5N1 virus escape mutants conferring resistance to monoclonal antibodies (MAbs) may correlate with a decrease of virus virulence for mice and that the virulence can be restored to the initial level by serial passages. In the present study, the mutations identical to those observed in the HA of a low-virulent escape mutant and its readapted variant were introduced into the HA gene by site-specific mutagenesis. The viruses produced by plasmid transfection and containing the HA gene either of A/Vietnam/1203/2004 (H5N1) virus with a deletion at the cleavage site, or of a low-virulent escape mutants, or of its readapted variant, in the presence of 6 genome segments of A/Puerto Rico/8/34 (H1N1) virus and the NA gene of A/Vietnam/1203/2004 (H5N1) virus, were assayed for virulence.

Postreassortment amino acid substitutions in influenza A viruses.

The genome of the influenza A virus consists of eight single-stranded negative sense RNA segments. Segmentation allows reassortment of genes between influenza A virus strains when two strains infect one host cell. Reassortment may lead to the emergence of pandemic influenza viruses.

Antigenic epitopes in the hemagglutinin of Qinghai-type influenza H5N1 virus.

The highly pathogenic avian influenza H5N1 viruses have become widespread and evolved into several clades. In our previous studies, the antigenic sites of the H5 hemagglutinin (HA) were characterized by selection and sequencing of escape mutants. In the present studies we analyzed the antigenic epitopes recognized by monoclonal antibodies against avian influenza A/Duck/Novosibirsk/56/05 (H5N1) virus isolated in western Siberia and belonging to subclade 2.

Readaptation of a low-virulence influenza H9 escape mutant in mice: the role of changes in hemagglutinin as revealed by site-specific mutagenesis.

In our earlier studies, we showed that an escape mutant of mouse-adapted H9N2 influenza virus carrying a T198N amino acid change in heamagglutinin (HA) has a lowered virulence for mice. The readaptation of this mutant to mice was associated with N198S or N198D reverse mutations. In this study, single-gene reassortants having HA gene of the wild-type virus, its low-virulence escape mutant, or a readapted variant were generated by site-specific mutagenesis and assayed for virulence.

[Amino acid substitutions in the hemagglutinin of H5 influenza virus changing the antigenic specificity and virus virulence].

In our earlier studies, we mapped the hemagglutinin antigenic epitopes of H5 influenza virus by selecting mutants resistant to the neutralizing effect of the antibody (escape mutants). Several escape mutants were shown to have a lowered virulence for mice. The readaptation of low-virulent escape mutants to mice resulted in the restoration of virulence.

Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule.

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies.

[Differential incorporation of genomic segments into the influenza A virus reassortants in mixed infection].

The gene composition of the viral population obtained via mixed infection of embryonated chick eggs with influenza viruses at a high multiplicity of infection was analyzed. In mixed infection caused by influenza A/WSN/33 (H1N1) and A/Duck/Czechoslovakia/56 (H4N6) viruses, the population showed a preponderance of the reassortants whose content of genomic segments originating from either of the parent virus deviated strongly from the random pattern: the hemagglutinin (HA) gene of A/WSN/33 (H1N1) virus and the NP gene of A/Duck/Czechoslovakia/56 (H4N6) virus were prevalent in the gene composition of the reassortants. The mixed infection produced by influenza A/Udorn/72 (H3N2) virus and the reassortant R8 containing the HA gene of A/Duck/Ho Chi Minh/014/78 (H5N3) virus, the population of reassortants contained mainly the HA gene of A/Udorn/72 (H3N2) virus and the NP gene of the reassortant R8.

Post-reassortment amino acid change in the hemagglutinin of a human-avian influenza H5N1 reassortant virus alters its antigenic specificity.

It was shown earlier that the reassortant influenza virus having hemagglutinin (HA) gene of A/Duck/Primorie/2621/2001 (H5N2) virus and 7 genes of A/Puerto Rico/8/34 (H1N1) virus produced low yields in embryonated chicken eggs. We found that a variant reassortant selected by serial passages in eggs produced higher yields than the initial reassortant. The variant reassortant had an amino acid substitution in the hemagglutinin N244D (H3 numbering).

Adjustment of receptor-binding and neuraminidase substrate specificities in avian-human reassortant influenza viruses.

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 alpha2-3-sialylated carbohydrate chains, while human virus HA interacts with alpha2-6 chains and low-active NA cleaves both alpha2-3- and alpha2-6-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.

Deviation from the random distribution pattern of influenza A virus gene segments in reassortants produced under non-selective conditions.

High-frequency reassortment of gene segments is characteristic for influenza viruses, and it is considered to be of significance for the origin of pandemic influenza. In order to analyze whether the segregation of genes in the reassortants is random, or it deviates from the random pattern, we inoculated embryonated chicken eggs simultaneously with two influenza viruses, A/WSN/33 (H1N1) and A/Duck/ Czechoslovakia/56 (H4N6), at a high multiplicity of infection. The virus yield was used for plaque cloning, and the genetic content of plaque isolates was determined by analysis of the mobility of virus-induced proteins in polyacrylamide gel (for NP and NS genes), partial sequencing (for M gene) and polymerase chain reaction analysis with strain-specific primers for the other genes.

[Optimization of the gene composition of influenza H5 virus hemagglutinin-containing reassortants and their efficacy in immune cross-protection experiments].

The reassortant described in the authors' previous paper contained 6 genes originating from the high-yield virus A/Puerto Rico/8/34 (H1N1) and the genes of hemagglutinin (HA) and neuraminidase (NA) of the low-pathogenic avian influenza A/Duck/Primorie/2621/2001 (H5N2) (6:2 reassortant). The reassortant was used for the backcrossing with the parent avian virus in order to optimize the gene composition. Genotyping of the highest-yield second-generation reassortment indicated that it had obtained the PB1, HA, and NA genes from the virus A/Duck/Primorie/ 2621/2001 and the other genes received the genes from the virus A/Puerto Rico/8/34 (5:3 reassortant).

Epitope mapping of the hemagglutinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies.

We mapped the hemagglutinin (HA) antigenic epitopes of a highly pathogenic H5N1 influenza virus on the three-dimensional HA structure by characterizing escape mutants of a recombinant virus containing A/Vietnam/1203/04 (H5N1) deltaHA and neuraminidase genes in the genetic background of A/Puerto Rico/8/34 (H1N1) virus. The mutants were selected with a panel of eight anti-HA monoclonal antibodies (MAbs), seven to A/Vietnam/1203/04 (H5N1) virus and one to A/Chicken/Pennsylvania/8125/83 (H5N2) virus, and the mutants' HA genes were sequenced. The amino acid changes suggested three MAb groups: four MAbs reacted with the complex epitope comprising parts of the antigenic site B of H3 HA and site Sa of H1 HA, two MAbs reacted with the epitope corresponding to the antigenic site A in H3 HA, and two MAbs displayed unusual behavior: each recognized amino acid changes at two widely separate antigenic sites.

[Reassortment and gene interactions in the crossing of low-pathogenic avian influenza H5 virus with human influenza virus].

The reassortants obtained via the crossing of highly productive influenza virus A/Puerto Rico/8/34 (H1N1) strain and the low pathogenic avian influenza virus A/Duck/Primorie/2621/2001 (H5N2) strain were genotyped and characterized. The H5N2 reassortant having 6 genes from A/Puerto Rico/8/34 virus has the high level of reproduction in chick embryos, while slightly more moderate than in the parent A/Puerto Rico/8/34 strain. The reproduction of the H5N1 reassortant that had 7 genes from A/Puerto Rico/8134 virus was very low.

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.