RESUMO
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.
Assuntos
Vírus da Influenza A Subtipo H5N2 , Vacinas contra Influenza , Vírus Reordenados , Temperatura , Vacinas Atenuadas , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Charadriiformes/imunologia , Embrião de Galinha , Galinhas/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Vírus da Influenza A Subtipo H5N2/genética , Vírus da Influenza A Subtipo H5N2/imunologia , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Influenza Aviária/prevenção & controle , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Camundongos , Modelos Animais , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Vacinação , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Replicação ViralRESUMO
The present study in BALB/c mice was conducted to compare immunogenicity and protective efficacy of several candidate vaccines based on homologous and heterologous strains after challenge with the highly pathogenic avian influenza strain A/Chicken/Kurgan/3/2005. The experimental vaccine composed of an inactivated split A/Vietnam/1194/2004 (H5N1) strain and a plant derived adjuvant has demonstrated better immunogenic properties versus the variant of the vaccine with aluminum hydroxide. Interestingly, the heterosubtypic H1N1 live attenuated vaccine candidate administered intranasally protected 93% of the subject against their challenge with HPIV HSN1.
Assuntos
Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Humanos , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/administração & dosagem , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Vírus Reordenados/imunologiaRESUMO
Analysis of the data of annual (1980-2005) monitorings of influenza A viruses in the North Caspian Sea basin and the Volga river delta, as well as the primary hemagglutinin structure of isolates of different years revealed the circulation of A/H13 and A/H16 viruses among gulls since 1976. Phylogenetic analysis revealed 3 significantly different evolutionary lines: an American line, a European line, and a line comprising the isolates from America and Eurasia. The H13N6 and H16N3 viruses isolated in Russia replicated in the respiratory and intestinal tracts of ducks and induced the production of antibodies; the H16N3 viruses induced the antibodies neutralizing viruses of subtype H16 only. The use of glycoconjugate polymers showed that the receptor phenotype of the study H16 viruses differed from that of the H13 viruses in its capacity to bind to 3'SL with a higher affinity than alphaNANA. The comparative phylogenetic analysis suggests the existence of the common precursor of H13 and H16 viruses and their further evolution in relation to environmental conditions, including their adaptation to a new host.
Assuntos
Charadriiformes/virologia , Vírus da Influenza A/classificação , Influenza Aviária/epidemiologia , Influenza Aviária/virologia , Substituição de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Patos/virologia , Monitoramento Ambiental , Monitoramento Epidemiológico , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Dados de Sequência Molecular , Testes de Neutralização , Filogenia , Receptores Virais/metabolismo , Federação Russa , Replicação ViralRESUMO
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.
Assuntos
Genoma Viral/genética , Vírus da Influenza A/genética , Filogenia , Análise de Sequência de DNA , Animais , Sequência de Bases , Embrião de Galinha , Patos/virologia , Humanos , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , MoscouRESUMO
The ability of influenza viruses from different hosts to bind to the intestinal epithelium of various birds (Anseriformes (Anatidae), Galliformes, Charadriiformes (sandpipers and sea gulls), Ciconiiformes (storks), Podicipediformes (grebes), and Gruiformes was studied. The composition of sialo-containing receptors on the epithelia was examined, by using lectins. Intestinal epitheliocytes of the Anatidae (Anseriformes) family was shown to have a low content of receptors binding both Sambucus nigra agglutinin (SNA) lectin specific to Siaalpha-6Gal, and Maackia amurensis agglutinin (MAA) lection specific to Siaalpha2-2Gal. Nevertheless, these cells well bound duck influenza viruses. The intestinal epithelium of Ciconiiformes, Podicipediformes, and Gruiformes well bound MMA lection, but avian influenza viruses weakly bound the latter. The intestinal cells of Gallinaceae bound both MMA and SNA lectins and avian and human influenza viruses. Thus, the composition of natural sialosides is different in various avian species whereas the receptor specificity of influenza viruses from various hosts reflects these differences. This can be accounted for by the differences in the ability of influenza viruses from different birds to break through the interspecies barrier, infecting mammals and human beings in particular.
Assuntos
Aves , Vírus da Influenza A/fisiologia , Influenza Aviária/virologia , Mucosa Intestinal/química , Mucosa Intestinal/metabolismo , Mucinas , Oligossacarídeos , Receptores Virais/química , Receptores Virais/metabolismo , Mucosa Respiratória/química , Mucosa Respiratória/metabolismo , Animais , Aves/classificação , Aves/virologia , Humanos , Lectinas , Macaca , Modelos Moleculares , Mucinas/análise , Oligossacarídeos/análise , Oligossacarídeos/metabolismo , Especificidade da EspécieRESUMO
The receptor properties of H1 and H2 influenza viruses (IV), isolated from duck, pig and man were studied by using the natural and synthetic sialoglycoconjugates. It was shown that viruses, isolated from different hosts, adapt themselves to the host cell receptors. The IV affinity was increasing to 6'sialy(N-acetyllactosamine) in proportion as amino acids (in positions 138, 190, 194 and 225), which are for avian IV, were increasingly replacing. Some of the porcine viruses display adaptation to the human receptor, i.e. 6'sialy(N-acetyllactosamine), however, all tested porcine influenza viruses, belonging to different evolution branches, acquired even more affinity to sulphated and fucozyled derivatives of 3'sialy(N-acetyliactosamine)-(Neu5AC alpha 2-3 g AL beta 1-4(fUC alpha 1-3)(6-sulfo)GlcNAc beta).
Assuntos
Evolução Biológica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Orthomyxoviridae/fisiologia , Receptores Virais/metabolismo , Adaptação Fisiológica , Aminoácidos/metabolismo , Amino Açúcares/química , Amino Açúcares/metabolismo , Animais , Patos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Modelos Moleculares , Orthomyxoviridae/imunologia , Orthomyxoviridae/isolamento & purificação , Especificidade da Espécie , Sulfatos , SuínosRESUMO
The receptor properties of influenza virus (IF) isolates/SSSR/90/77 are studied. The isolates are peculiar for losing glycosylation sites (GS) at the Asn131 receptor-binding region (GS131) after passaging in mice and at the Asn158 region (GS158) after cultivation in the presence of mouse serum. The loss of each carbohydrate residue increases the influenza virus affinity for carbohydrate chains with the terminal group Neu5Ac alpha 2-6Gal and reduces its affinity for Neu5Ac alpha 2-3Gal receptors. The effect is more pronounced in the GS158-depleted virus. Upon substitution of asparagine by aspartic acid, the electrostatic component of virus binding to the receptor is altered because of the increased negative charge on hemagglutinin. The virus receptor phenotype changes depending on the cultivation conditions. The isolate adapted to mice has higher affinity to mouse lung cell receptors, while the virus propagated in chick embryos in the presence of inhibitors has higher affinity to allantoic membrane cells.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A/fisiologia , Receptores de Superfície Celular/metabolismo , Animais , Asparagina/metabolismo , Sítios de Ligação , Metabolismo dos Carboidratos , Membrana Celular/virologia , Embrião de Galinha/virologia , Glicosilação , Hemaglutininas/metabolismo , Vírus da Influenza A/patogenicidade , Pulmão/metabolismo , Pulmão/virologia , Camundongos , Mutação , Ácido N-Acetilneuramínico/metabolismo , Fenótipo , Eletricidade EstáticaRESUMO
The affinity of the duck, chicken, and human influenza viruses to the host cell sialosides was determined, and considerable distinctions between duck and chicken viruses were found. Duck viruses bind to a wide range of sialosides, including the short-stem gangliosides. Most of the chicken viruses, like human ones, lose the ability to bind these gangliosides, which strictly correlates with the appearance of carbohydrate at position 158-160. The affinity of the chicken viruses to sialoglycoconjugates of chicken intestine as well as chicken, monkey, and human respiratory epithelial cells exceeds that of the duck viruses. The human influenza viruses have high affinity to the same cells but do not bind at all to the duck epithelial cell. This testifies to the absence of 6'-sialylgalactose residues from the duck cells, in contrast to chicken and monkey cells. The alteration of the receptor specificity of chicken viruses in comparison with duck ones results in the similarity of the patterns of accessible cells for chicken and human influenza viruses. This may be the cause of the appearance of the line of H9N2 viruses from Hong Kong live bird markets with receptor specificity similar to that of H3N2 human viruses, and of the ability of H5N1 and H9N2 chicken influenza viruses to infect humans.
Assuntos
Vírus da Influenza A/fisiologia , Receptores de Superfície Celular/metabolismo , Animais , Sequência de Carboidratos , Galinhas , Patos , Células Epiteliais/virologia , Gangliosídeos/metabolismo , Humanos , Vírus da Influenza A/patogenicidade , Dados de Sequência Molecular , Doenças das Aves Domésticas/virologia , Especificidade da EspécieRESUMO
Neoglycoconjugates containing 4, 8, 16, 32, and 64 terminal residues of B-disaccharide (BDI) or N-acetylneuraminic acid (Neu5Ac) attached to poly(aminoamide)-type dendrimers (PAMAMs) were synthesized. The ability of BDI conjugates to bind natural xenoantibodies (anti-BDI antibodies) and the ability of Neu5Ac conjugates to inhibit the hemagglutinin-mediated adhesion of influenza virus were studied. The biological activity of PAMAM conjugates turned out to be higher than that of free carbohydrate ligands, but less than that of multivalent glycoconjugates based on other types of synthetic polymeric carriers. A conformational analysis of PAMAM matrices and resulting conjugates was performed to determine the statistical distances between carbohydrate ligands. The computations revealed the tendency of the PAMAM chains toward compaction and formation of dense globules. The process results in a decrease in the distances between the carbohydrate ligands in the conjugates and, hence, could affect the ability of glycoconjugates to efficiently bind the polyvalent carbohydrate-recognizing proteins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 6; see also http://www.maik.ru.
Assuntos
Antivirais/síntese química , Glicoconjugados/síntese química , Imunossupressores/síntese química , Poliaminas/química , Anticorpos Heterófilos/sangue , Anticorpos Heterófilos/imunologia , Antivirais/química , Antivirais/farmacologia , Dendrímeros , Dissacaridases/química , Glicoconjugados/química , Glicoconjugados/farmacologia , Humanos , Imunossupressores/química , Imunossupressores/farmacologia , Ligantes , Conformação Molecular , Estrutura Molecular , Ácido N-Acetilneuramínico/química , Orthomyxoviridae/efeitos dos fármacosRESUMO
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.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/enzimologia , Vírus da Influenza A/metabolismo , Neuraminidase/metabolismo , Vírus Reordenados/enzimologia , Vírus Reordenados/metabolismo , Vírus da Influenza A/genética , Vírus Reordenados/genética , Especificidade por SubstratoRESUMO
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.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Vírus da Influenza A/fisiologia , Neuraminidase/fisiologia , Replicação Viral/genética , Animais , Linhagem Celular , Meios de Cultura , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/enzimologia , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Rim/ultraestrutura , Rim/virologia , Microscopia Eletrônica , Neuraminidase/genética , Virulência/genéticaAssuntos
Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/prevenção & controle , Vacinas Virais/imunologia , Adolescente , Adulto , Animais , Anticorpos Antivirais/sangue , Relação Dose-Resposta Imunológica , Avaliação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunização , Camundongos , Fatores de Tempo , Ultrafiltração , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversos , Vacinas Virais/isolamento & purificação , Cultura de Vírus/métodosRESUMO
The contact sites of yeast tRNA1Val with tRNA(adenine-1-)-methyltransferase (EC 2.1.1.36) were studied by comparing the partial digests of free and enzyme bound tRNA. The RNAases Sa, V1, S1 and A were used for this purpose. Phosphodiester bonds on the proximal end of the acceptor stem and adjacent D- and T psi-stems, forming a continuous area, are protected by the methylase from the action of RNAases. On the contrary an enhancement of phosphodiester bond splitting of the D- and T psi-loops and of the anticodon stem is observed in the presence of methylase, which is interpreted as tertiary structure relaxation of tRNA owing to complex formation. The isotherm of ethidium bromide adsorption on free and methylase bound tRNA has shown that in enzyme shields approximately 50% of tRNA double stranded regions.
Assuntos
RNA de Transferência Aminoácido-Específico/metabolismo , RNA de Transferência de Valina/metabolismo , Thermus/enzimologia , tRNA Metiltransferases/metabolismo , Sítios de Ligação , Ácido Edético , Etídio , Hidrólise , Substâncias Macromoleculares , RNA Fúngico/metabolismo , Ribonucleases , Análise EspectralRESUMO
tRNA(adenine-1-)-methyltransferase (EC 2.1.1.36) was isolated from the extreme thermophile Thermus thermophilus strain HB8. The specific activity of the enzyme is about 50 000 and the yield of activity more than 20%. The method of isolation consists of five steps and is valid for isolation of mg quantities of the enzyme. The purified protein preparation is practically homogeneous in SDS-gel electrophoresis, the position of the protein band corresponds to a molecular weight of 25 000. By gel filtration on Sephadex G-100 the molecular weight of the native protein was found to be 70 000. These data allow to suggest a subunit structure of the enzyme. The enzyme is highly thermostable and is most active at 80 degrees C. The only activity of the enzyme is to methylate A58 in the T psi X loop of tRNA.
Assuntos
Thermus/enzimologia , tRNA Metiltransferases/isolamento & purificação , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Peso MolecularRESUMO
A new type of sorbents for affinity chromatography is suggested and used to purify tRNA methylases. tRNA was immobilized on aminooxybutylcellulose via the oxidized 3'-end. In order to bind other enzymes specific for nucleic acids in general, e. g. nucleases, and to achieve a higher degree of purification the crude enzyme preparation was treated with rRNA immobilized on aminooxybutycellulose. The sequential application of two sorbents mentioned allows to get an approximately two hundred fold purification of total tRNA methylases. In a separate experiment the possibility of individual tRNA methylase fractionation by means of elution with a NACl gradient was shown; the degree of purification for some methylases was more than a thousand fold.