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.

[The generation and characteristics of reassortant influenza A virus with H5 hemagglutinin and other genes from the apathogenic virus H6N2].

The experimental reassortant vaccine strain VN-gull (H5N2) containing H5 hemagglutinin (HA) with a removed polybasic site in the connecting peptide and other genes from the apathogenic H6N2 virus A/gull/Moscow/3100/2006 (gull/M) was obtained using a two-step protocol. At Step 1, the reassortant with HA of A/Vietnam/1203/04-PR8/ CDC-RG and other genes from cold-adapted A/Leningrad/17/47 (VN-Len) viruses was generated due to selection with antibody to H2N2 at 26 degrees C. At Step 2, the reassortant VN-gull was obtained by replacing all genes from Len with those from gull/M due to selection with antibody to H6N2 at 39 degrees C. The reassortant VN-Len was apathogenic and the reassortant VN-gull was weakly virulent in mice. Both gave rise to specific antibodies and 4 weeks after single inoculation they provided complete protection against further challenge with highly pathogenic HSN1 virus A/chicken/Kurgan/3/05 (H5N1) (Ku-Len). The chickens infected with live VN-gull virus showed neither clinical symptoms, nor fecal virus excretion; nevertheless, they gave rise to antibodies and were protected from the further challenge with A/chicken/Kurgan/3/2005. The high yield, safety, and protectivity of VN-Len and Ku-Len made them promising strains for the production of inactivated and live vaccines against H5N1 viruses.

[The study of the nonpathogenic influenza virus A/gull/Moscow/3100/2006 (H6N2) isolated in Moscow].

The influenza virus A/gull/Moscow/3100/2006 (H6N2) was isolated from gull feces within the precincts of Moscow in autumn 2006. The nucleotide sequence of the complete genome (GenBank, EU152234-EU152241) and genotype (K, G, D, 6B, F, 2D, F, 1E) for this virus were determined. Phylogenetic analysis suggests that the H6N2 virus derived by numerous reassortment between viruses that have been circulating among different birds in Europe since 1999 and in South-East Asia (NA gene) for last years. Migratory birds probably introduced some of these viruses from South-East Asia earlier. The strain A/gull/Moscow/3100/2006 is nonpathogenic for chicken embryos and mice and induces specific antibody production in mice. Similar to all avian influenza viruses A/gull/Moscow/3100/ 2006 it binds to Neu5Ac(2-3Gal receptors, but reveals higher affinity for fucosylated sialosugars (SLex) in contrast to the duck viruses, as was shown in receptor specificity assay and clarified due to modeling the accommodation of SLex into receptor binding site of duck and gull influenza virus hemagglutinin.

[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.

[Reduction of the functional match of influenza virus hemagglutinin and neuraminidase after reassortation of genes]. / Vosstanovlenie funktsional'nogo sootvetstviia gemaggliutinina i neiraminidazy virusa grippa posle reassortatsii genov.

Influenza virus A (FluA) reassortants with low-functional neuraminidase (NA) of subtype N1 and hemagglutinin (HA) of subtypes H2, H3, H4, and H13 display virion aggregation and accumulate to a lower titer because sialyl residues are not completely removed from virion components. Nonaggregating variants of FluA (H13N1) were shown to result from a mutation that reduces the HA affinity for sialyl substrates. Amino acid substitution K156E, which increases a negative charge at the edge of the receptor-binding pocket of HA large subunit (HA1), was revealed in two independent variants. This substitution was the only difference between HA1 of the original reassortant and one of its variants and, therefore, accounted for restoration of the functional match between HA and NA.

[Gene analysis and phenotypic characteristic of highly-reproductive reassortants, containing the gene for bird influenza virus subtype H2 hemagglutinin]. / Gennyi analiz i fenotipicheskaia kharakteristika vysokoreproduktivnykh reassortantov, soderzhashchikh gen gemaggliutinina virusa grippa ptits podtipa H2.

A series of reassortant clones with antigenic formulae H2N1 and H2N3 were produced by genetic reassortment performed with the use of an avian influenza virus, A/Pintail Duck/Primorie/695/76 (H2N3) and a high-yield reassortant strain X-67. Preliminary identification of the parent origin of NP and NS genes for 5 reassortants was performed by comparison of the mobilities of virus-specific proteins in polyacrylamide gel electrophoresis. The parent origin of genes of internal and nonstructural proteins for 3 reassortants was identified by partial sequencing. Although the genes of internal and nonstructural proteins of the reassortants originated from high-yield X-67 virus, only H2N3 reassortants were similar to the high-yield parent virus as concerns the level of the virus accumulation evaluated by hemagglutination titration and measurement of the virus protein content.

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.

Postreassortment changes in influenza A virus hemagglutinin restoring HA-NA functional match.

An important function of influenza virus neuraminidase (NA) is the removal of sialic acid residues from virion components in order to prevent the aggregation of virus particles. In previous communications we have reported that reassortant viruses containing the NA gene of A/USSR/90/77 (H1N1) virus and HA genes of H3, H4, H10, or H13 subtypes had a tendency to virion aggregation at 4 degrees C and that the virion clusters irreversibly dissociated after the treatment with bacterial neuraminidase. It was concluded that in such reassortants the removal of sialic acid residues is inefficient. Nonaggregating variants of the reassortants were selected in the course of serial passages in embryonated chicken eggs. In the present paper a reassortant virus, R2, having the HA gene of A/Duck/Ukraine/1/63 (H3N8) virus and the other genes of A/USSR/90/77 (H1N1) virus, as well as its non-aggregating passage variants and both parent viruses, have been studied in order to reveal the presence of unremoved sialic acid residues in the virions. An assay of sialic acid content by high-performance liquid chromatography with fluorescent detection has revealed the presence of sialic acid in the purified virus preparations of A/USSR/90/77 (H1N1) virus and the R2 reassortant and its nonaggregating variants, whereas only trace amounts of sialic acid have been detected in the A/Duck/Ukraine/1/63 (H3N8) parent virus. The data obtained with the use of the labeled "indicator" virus suggest that the unremoved sialic acid residues are present at the virion surface. The nonaggregating variants have been shown to possess a lower affinity toward high-molecular-weight sialic acid-containing substrates compared to the initial reassortant R2. Sequencing of HA genes has revealed amino acid changes in the nonaggregating variants compared to the initial reassortant. One substitution, N248D in HA1, is the same in two independently selected nonaggregating variants. The presented data suggest that the complete removal of sialic acid residues by viral NA from the virion components is not obligatory for the absence of virus particle aggregation: the latter may be achieved (in the reassortants and, presumably, in the wild-type virus) through a balance between the degree of HA affinity toward the sialic acid-containing receptors and the extent of the removal of sialic acid residues by NA.

[A mechanism for limiting reproduction influenza A virus reassortants with incomplete functional compatibility of hemagglutinin and neuraminidase gene products]. / Mekhanizm ogranicheniia reproduktsii reassortantov virusa grippa A pri nepolnom gunktsional'nom sootvetstvii produktov genov gemaggliutinina i neiraminidazy.

The mechanism of decrease in the level of virus accumulation in reassortants with hemagglutinin (HA) and neuraminidase (NA) genes from different parents is studied. The reassortant viruses and their passage variants do not differ by the rate of virus protein production or their stability in infected cells. Electron microscopy and titration of infectious virus in culture fluid and cell-associated virus showed that the variants selected by serial passages accumulated mainly in the culture fluid, whereas the initial reassortant virions were predominantly cell-associated. These data suggest that incomplete removal of sialic acid residues by viral neuraminidase N1 in some reassortants results in re-attachment of virions to the infected cells and thus impairs the virus dissemination, which may be regarded as a reassortant-limiting factor probably significant for virus evolution.