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. Continuously evolving highly pathogenic influenza A viruses are a serious health concern which necessitates a coupling of theoretical and experimental approaches to predict the evolutionary trends of the influenza A virus, with a focus on the H5 subtype.

[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. As it has been found the substitutions appeared in the HA2 of m4G(10) and m4G(6) viruses compensated negative effect of the S145P1 mutation and provided a significant increase in the viral replication ability at the early stage of infection in embryonated chicken eggs as well as in HA thermostability in comparison with m4F11(4) mutant. Phenotypic properties that provide advantages in the process of virus replication can play a role of the positive selection factor in viral population.

[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. 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.

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

KEYWORDS: influenza virus; reassortment; virus yield; gene constellation.

[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. High immunogenicity of ReM8 reassortant was demonstrated by immune protection studies in mice. The reassortant virus ReM8 is suitable as a candidate strain for the production of inactivated and subunit influenza vaccines.

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. The results showed that antibody-selected mutations in the HA of H9 influenza virus can decrease mortality and virus accumulation in mouse lungs, though not in nasal turbinates, and the effect may be compensated by reverse mutations in the course of passaging.

[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. Determination of virulence for mice indicated that amino acid substitution in the HA gene of a low-virulent escape mutant produced a decrease of virulence whereas the additional mutation identical to that acquired by the escape mutant in the course of readaptation restored the virulence to initial level. The findings are the first strong evidence for lower H5N1 virus virulence resulting from the amino acid substitution changing the antigenic specificity of HA and for restored virulence arising from compensating mutation in the HA gene.

[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. In the present communication. We present data on the assay of virulence of single-gene reassortants containing HA genes of the wild-type virus, low-virulent escape mutant, or re-adapted variant, and the other genes of a mouse-adapted H9N2 Influenza virus. The results demonstrate that the amino acid change S145F (H3 numbering) in the hemagglutinin ensuring the resistance to a monoclonal antibody can be deleterious to virulence, and that the damaging effect on virulence may be compensated for by additional amino acid changes in position 186 in the hemagglutinin arising in the course of virus passaging in mice. The data indicate that the compensational mutations restoring the pathogenic potential of antigenic variants may be regarded as an additional factor in the evolution of influenza virus hemagglutinin.

[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. The findings are discussed due to the problem of specific recognition of gene segments when incorporated into the viral particles.

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). In this report we demonstrated that the post-reassortment amino acid substitution N244D altered the antigenic specificity of HA as revealed by the loss of reactivity with an anti-H5 monoclonal antibody in hemagglutination-inhibition (HI) test. The results are discussed in association with the evolution of H5 hemagglutinin.

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. Out of 37 isolates, 27 were reassortants. The majority of the reassortants contained the HA gene of A/WSN/33 (H1N1) virus and the NP gene of A/Duck/Czechoslovakia/56 (H4N6) virus. The data demonstrate the previously unrecognized phenomenon of segment-specific deviation from the random distribution of parent genes in the reassortants. The results are discussed in connection with the problem of differential competition between influenza A virus gene segments in mixed infection and random versus non-random reassortment of gene segments under non-selective conditions.

[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). The yield produced in the embryonated chicken eggs by the 5:3 reassortant was higher than that produced by the 6:2 reassortant although it did not achieve the reproduction of the parent virus A/Puerto Rico/8/34. Murine immunization with the inactivated reassortant containing the HA and NA genes of the virus A/Duck/Primorie/2621/2001 (H5N2) provided an efficient protection against the virus containing HA and NA of a recent H5N1 strain.

[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. The serial passage selection allowed the investigators to obtain the H5N1 strain that was reproductively close to the H5N2 reassortant. This variant had one amino acid substitution in hemagglutinin (N244D, H3 numbering) and a lower affinity for fetuin. By the level of virulence to mice, the H5N1 and H5N2 reassortants were close to A/Puerto Rico/8/34 virus and greatly differed in this respect from low virulent A/Duck/Primorie/2621/2001 (H5N2). The results are discussed in connection with the problem of vaccination when there is a threat for H5N1 virus subtype-caused pandemic.

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.

[Evolution of avian influenza viruses H5N1 (1997-2004) in southern and south-eastern Asia].

Highly pathogenic avian influenza viruses of the H5N1 subtype are widespread and have become endemic in poultry in southern and southeastern Asia. An unprecedented epizootic was caused by these viruses in 8 countries in the winter of 2003 to 2004. This fact along with more frequent human cases of the infection with unusually high mortality rates in Vietnam and Thailand raises concern that these H5N1 events may lead to a new influenza A virus pandemic. This review summarizes the results of studies dealing with the ecology and evolution of avian influenza H5N1 viruses in southern and southeastern Asia since 1997. The pathogenesis of the infection in human beings and laboratory animals and possible determinants of the high pathogenicity of H5N1 viruses in mammals are considered. A scheme for designing modified H5N1 vaccines using the latest advances in reverse genetics of influenza viruses is given.

Postreassortment changes in a model system: HA-NA adjustment in an H3N2 avian-human reassortant influenza virus.

In our previous studies we described the postreassortment changes in the hemagglutinin (HA) of H2N1, H3N1, H4N1 and H13N1 influenza A virus reassortants with HAs derived from avian viruses and low-functional neuraminidase (NA) of a human parent virus A/USSR/90/77 (H1N1). The changes involved amino acid substitutions that increased the negative local charge in the vicinity of the receptor-binding pocket and decreased the affinity of HA to sialic acid receptors. In the present report we describe the studies performed with H3N2 reassortant viruses having HA of A/Duck/Ukraine/1/63 (H3N8) virus and NA of A/Aichi/2/68 (H3N2) virus. Amino acid changes in the HA gene registered in virus variants selected in the course of serial passages resulted in a decrease in the affinity to sialic acid-containing substrates and cell receptors. However, the decrease was less expressed than in the reassortants containing the low-functional NA of N1 subtype described in our earlier studies, and the amino acid changes were not necessarily associated with an increase of negative charge. In one passage variant an amino acid substitution in NA was detected. The relevance of these results for the evolution of the H3N2 virus of the 1968 pandemic is discussed.

Antigenically relevant amino acid positions as revealed by reactions of monoclonal antibodies with the nucleoproteins of closely related influenza A virus strains.

The nucleoproteins (NP) of a group of influenza A virus strains were analyzed with the use of a panel of anti-NP monoclonal antibodies (Mabs) in the radioimmunosorbtion reaction with subsequent polyacrylamide gel electrophoresis of the immune complexes, and in the immunoblotting. The group included 2 pairs of closely related viruses having minimal amino acid differences in NP sequence. The analysis of the results of the immune reactions in comparison with the known amino acid sequences of NP allowed us to suggest that the Mab 150/4 recognizes the epitope containing amino acid residues in positions 196 and/or 290, whereas the amino acid residue in position 353 participates in the formation of the antigenic epitope reacting with the Mab 7/3.

[Variation of primary structure and conformation of NP protein from influenza A virus, recognized by monoclonal antibodies]. / Variatsii pervichnoi struktury i konformatsii belka NP virusa grippa A, razpoznavaemy monoklonal'nymi antitelami.

The antigenic structure of influenza A NP protein was studied by using a panel of 12 monoclonal antibodies (mAbs). A group of strains of influenza-A virus with the known NP amino acid sequence was analyzed by radioimmunoprecipitation with a subsequent analysis of the immune complexes by electrophoresis in polyacrylamide gel. The examined group of strains comprised pairs of closely related variants like A/USSR/90/77 and A/Brazil/1/78 as well as A/Puerto Rico/8/34 (Mount Sinai variant) and A Puerto Rico/8/34 Cambridge 1 variant). The results made it possible to identify position 353 as a part of antigenic site. The combined results of radioimmunoprecipitation and immunoblotting suggest that the mentioned site is recognized by mAb IVE8 as a linear epitope and by mAb as a confirmation epitope. Amino acids in positions 196 and/or 290 are involved in the formation of another antigenic site. The N-terminal part of NP, removed in the course of protein processing, is not involved in the differentiation of avian and human strains by mAb 315. None of the mAbs used in our study recognized the N-terminal part of NP as a linear epitope.

[Interspecies transmission of influenza A viruses and influenza pandemics]. / Mezhvidovaia transmissiia virusov grippa A i problema pandemii.

Molecular and genetic data are summarized on the origin of influenza A virus pandemic variants. Conceptual modifications of the reassortment theory of the origin of pandemic strains are discussed in connection with the appearance of new H5 and H9 avian influenza viruses, which caused the respiratory infection in man and which are presently in the focus of attention as possible agents of future pandemic.

[Functional interactions of the influenza virus glycoproteins]. / Funktsional'noe vzaimodeistvie glikoproteinov virusa grippa.

Hemagglutinin (HA) and neuraminidase (NA) are functionally related coat glycoproteins of the influenza virus (Flu). HA interacts with terminal sialyl residues of oligosaccharides and ensures the binding of the virus particle to the cell surface. NA is a receptor-destroying enzyme that removes sialyl residues from oligosaccharides contained in cell and virus components and thereby prevents aggregation of virus particles. Analysis of reasortants combining low-functional NA of human Flu with HA of avian Flu showed that sialyl residues are not completely removed in some cases. With high HA affinity for sialyl substrates, such virus particles aggregate, aggregates accumulate on the cell surface, and virus yield decreases. Serial passaging of low-yield aggregating reassortants may lead to selection of high-yield variants, which do not aggregate. A loss of aggregation is due to a decrease in HA affinity for high-molecular-weight sialyl substrates. On evidence of sequencing of the HA gene in original reassortants and their nonaggregating variants, HA affinity is reduced and aggregation lost owing to a mechanism common for different HA antigenic subtypes (H2, H3, H4, and H13). This is an increase in the negative charge as a result of an amino acid substitution in the vicinity of the receptor-binding pocket of HA. Taken together, these findings suggest a way of postreassortment adaptation which improves the functional match of HA and NA. The experimental system employed provides a model of natural processes associated with generation of Flu variants having a pandemic potential.