[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.

[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.

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.

The immunosorbtion of influenza virus nucleocapsids from cell lysates as a technique for the studies on viral RNA synthesis.

Radioimmunosorbtion of influenza virus nucleocapsids from lysates of the infected cells was applied for studies on virus-specific RNA synthesis. The method allowed the isolation of intact-labelled viral RNA segments. The procedure included preadsorbtion of antiviral antibodies on protein A containing sorbents. The protein A containing sorbent with attached antibodies was mixed with lysates of influenza virus-infected [3H]uridine-labelled cells. Viral nucleocapsids bound by the antibodies to the sorbent were used for RNA extraction. The isolated RNA was analysed by polyacrylamide gel electrophoresis with subsequent autoradiography. The method allows the isolation of nondegraded labelled virus-specific RNA by means of a relatively simple procedure.

Newcastle disease virus-specific RNA: an analysis of 24 S and 35 S RNA transcripts.

The denaturation of Newcastle disease virus-specific 24 S and 35 S RNA by heat or formamide treatment led to a shift of a large part (60--80%) of RNA into the 18 S zone. The remaining 20--40% could not be dissociated further by repeated denaturation or by centrifugation in dimethyl sulfoxide-sucrose gradient. Hybridization-competition analysis revealed that the majority (approximately 75%) of the non-dissociable 35 S RNA and almost all the material present in the non-dissociable 24 S RNA were represented by nucleotide sequences homologous to 18 S RNA. On the other hand, the non-dissociable 35 S RNA lacked some of the sequences present in 18 S RNA, since no more than 45% of the labelled 18 S RNA could be displaced from the hybrid by an excess of unlabelled non-dissociable 35 S RNA. The possible origin of 24 S and 35 S RNA is discussed.

Newcastle disease virus-specific RNA: hybridization-competition of the non-dissociable 35 S RNA with individual 18 S RNA species.

The non-dissociable fraction of Newcastle disease virus- (NDV-Y specific 35 S RNA competed efficiently with two individual 18 S RNA transcripts in hybridization-competition experiments with 50 S virion RNA. The third 18 S component could not be displaced from the hybrid by an excess of non-dissociable 35 S RNA.

RNA sequences of opposite polarities transcribed from a large part of vaccinia virus genome are accumulated in the cytoplasm.

Labelled vaccinia virus DNA was used in saturation-hybridization experiments with RNA extracted from virus-infected cells. An excess of "late" cytoplasmic RNA converted 45% of DNA into DNA-RNA hybrids, whereas 17% of DNA could be converted into hybrids by RNA extracted from purified nuclei. RNA-RNA hybrids obtained from cytoplasmic RNA by self-annealing and ribonuclease treatment, were melted and used in hybridization with DNA: 36% of DNA was hybridized at the maximal concentration used.

[Reverse transcription of influenza virion RNA without exogenous primer]. / Obratnaia transkriptsiia virionnoi RNK virusa grippa bez vneshnei zatravki.

Reverse transcriptase has been shown to transcribe virion DNA of influenza A virus without an exogeneous primer. At least six virion RNA segments are transcribed with the formation of complementary 4S DNA product. A possible primer function of hairpin structures at the 3'-end of virion RNA segments is discussed.

[Molecular composition of phenotypically mixed virions formed during mixed infection with influenza viruses A and B]. / Molekuliarnyi sostav fenotipicheski smeshannykh virionov, formiruiushchikhsia pri dvoinoi infektsii, vyzvannoi virusami grippa A i B.

Upon mixed infection of cultured cells by influenza viruses A/WSN/33 and B/Lee/40 the produced virions contain in their envelopes either hemagglutinin B/Lee/40, or hemagglutinins of both viruses, depending on their concentration ratio during infection. In the first case the population contains RNA segments and nucleoproteins (NP) of both viruses, in the second-exclusively RNA and NP of virus A/WSN/33. Results of immunoprecipitation with monoclonal antibodies to protein of virus A/WSN/33 with further analysis of immunoprecipitates by electrophoresis in polyacrylamide gels did not reveal the presence of virus ribonucleoproteins, containing NP of both viruses. The data obtained demonstrate the high of specificity of protein-protein recognition during reassembly of virion inner structures.

[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.

[The mechanism of RNA synthesis in the influenza virus]. / Mekhanizm sinteza RNK virusa grippa.

The influenza virus RNA replication and transcription have been extensively studied both in infected cell and in vitro systems. The review presents the analysis of experimental data on the molecular mechanisms for influenza virus RNA replication and transcription as well as on the regulation of the synthesis of three kinds of virus-specific RNAs (vRNA, cRNA and mRNA) and their interrelationships. A hypothetical pattern of regulation is presented, providing an explanation for change in the rate of synthesis of RNA segments and their transcripts in the course of the replication cycle.

[Structural organization of the genome of paramyxoviruses]. / Strukturnaia organizatsiia genoma paramiksovirusov.

The review summarizes the recent papers on the studies of primary structure of genome of a number of paramyxoviruses from the three genera of a family. The cited data demonstrate that despite the common principles of the genetic material arrangement shared by paramyxoviruses, they are variable in the genome, the primary structure of intragenic region, as well as the strategy of coding for some proteins. The data on the arrangement of the genetic material is discussed as useful as a criterion for classification of single stranded viruses with unsegmented genome.

[Regulation of synthesis of virus-specific RNA in cells infected with influenza virus]. / Reguliatsiia sinteza virusspetsificheskikh RNK v kletkakh, zarazhennykh virusom grippa.

Interrelationships between the level of protein synthesis and the pattern of virus-specific transcription in influenza virus-infected cells have been studied with the use of a wide range of concentrations of a protein synthesis inhibitor (cycloheximide). The obtained data reveal a predominant stimulation of the transcription for some viral genes by partial suppression of protein synthesis. The data also suggest the existence of cRNA (full transcripts) synthesis regulation, in addition to the regulation of vRNA and mRNA synthesis described earlier.

[Synthesis of virus-specific RNA in cells infected with influenza B virus]. / Sintez virusspetsificheskikh RNK v kletkakh, zarazhennykh virusom grippa B.

Synthesis of virus-specific RNA in cells infected with influenza B virus was studied by means of PAGE analysis of RNA hybrid duplexes or nucleocapsid-associated RNA. A switch from an "early" to "late" pattern was registered in the relative rates of synthesis of the corresponding mRNA as well as in the synthesis of vRNA segments 7 and 8. Contrary to the pattern described earlier for influenza A virus, the relative rate of synthesis of mRNA 5 was increased at the late stage of the replication cycle; besides, the efficiency of transcription of the polymerase genes was higher. Partial suppression of protein synthesis with moderate concentration of cycloheximide at a late stage of infection resulted in a differential inhibition of vRNA segments synthesis and stimulation of mRNA synthesis, leading to restoration of an "early" pattern in both cases. The results confirm the general outline of regulation as presented for influenza A virus in an earlier publication and reveal several peculiarities in regulation of influenza B viral RNA synthesis.

[The use of immunosorption for the analysis of influenza virus RNA in intracellular viral ribonucleoproteins]. / Ispol'zovanie immunosorbtsii dlia analiza RNK virusa grippa, soderzhashcheisia vo vnutrikletochnykh virusnykh ribonukleoproteidakh.

Protein A-containing formaldehyde-fixed S. aureus (strain Cowan) was incubated with an antiviral serum or with a monospecific serum against NP protein, washed, and used as immunosorbent in order to isolate viral ribonucleoproteins (nucleocapsids) containing intact viral RNA from the extracts of influenza virus infected [3H]-uridine-labelled cells.

[The role of polymerase protein genes of influenza A virus in the transition from the early to late stage of replication]. / Rol' genov polimeraznykh belkov virusa grippa A v perekhode ot rannei stadii replikatsii k pozdnei.

Regulation of influenza virus RNA replication was studied with the use of A/FPV/Rostock/34 strain ts-mutants. Mutants C44, C15, C45 possessing the ts-defects in the PB2, PB1 and PA genes respectively were used for the infection of chick embryo cultured cells and H-uridine-labelled nucleocapsid-associated RNA was analysed in polyacrylamide gel electrophoresis to assess the kinetics of vRNA synthesis. A typical early-late transition of the pattern of vRNA synthesis was observed in the cells infected with C44, whereas the other two mutants exhibited a slightly changed (C15) or strongly distorted (C45) pattern. In shift up experiments after the transfer to non-permissive temperature all the mutants exhibited partial reversion to an early pattern of vRNA synthesis. The results are discussed in connection with the mechanism of the early-late transition of influenza virus-specific RNA synthesis.

[Analysis of the reassorted influenza virus clone genome: data for use of ordered selection of RNA segments]. / Analiz genoma reassortantnykh klonov virusa grippa: dannye v pol'zu uporiadochennogo podbora RNK-segmentov.

Chicken embryonated eggs were coinfected with influenza A/FPV/Rostock and A/FPV/Weybridge strains. 25 plaque isolates were obtained from the mixed yield and their genetic content was analysed by polyacrylamide gel electrophoresis of H3-uridine-labelled vRNA in a modified gel system. At least 18 clones out of 25 plaque isolates proved to be reassortants; however, only one among them contained the homologous RNA-segments belonging to both parents. The results are in agreement with the concept of an ordered recruitment of vRNA-segments into virions.

[Antigenic structure of influenza A virus subtype H5 hemagglutinin: mechanism of acquiring stability to monoclonal antibodies in escape-mutants]. / Antigennaia struktura gemaggliutinina virusa grippa A podtipa H5: mekhanizm priobreteniia ustoichivosti k monoklonal'nym antitelam u Escape-mutantov.

The analysis of escape mutants of the avian influenza virus of H5 subtype (strain A/Mallard/Pennsylvania/10218/84) revealed the location and structure of two antigenic sites in the hemagglutinin (HA) molecule. Several escape mutants exhibited unusual features in the reactions with monoclonal antibodies (Mabs), being completely resistant in the infectivity neutralization test to the Mabs used for their selection, and retaining the ability to bind the Mabs as revealed by enzyme-linked immunosorbent assay. An enhancement of the binding by an amino acid change in a different antigenic site was demonstrated, as well as a complete abolishment of the binding by a mutation selected by passage in the presence of an excess of the non-neutralizing Mab of high binding ability. The observed effects did not result from the changes in the affinity of the mutant HA toward sialic receptors. The data suggest that one amino acid change in HA may prevent the virus neutralization by different mechanisms for different Mabs: either the binding of the Mab to HA is prevented, or the bound Mab is unable to block the receptor-binding pocket of HA. Different mechanisms of the acquisition of resistance to Mabs in the course of the selection of escape mutants are discussed.