RESUMO
AIM: Study of ts, ca, att phenotype, immunogenicity and protective effectiveness of reassortants obtained by a way of recombination of a new influenza cold-adapted (ca) strain donor of attenuation A/Krasnodar/101/35/59 (H2N2) and virulent strain of influenza virus. MATERIALS AND METHODS: Viruses were used: ca strain A/Krasnodar/101/35.59 (H2N2), virulent strains: A/Kumamoto/102/02 (H3N2) and A/Bern/07/95. For determination of ts and ca phenotype, titration of viruses in chicken embryos was carried out simultaneously at optimal, decreased and increased temperature. Protective effect of immunization was evaluated during intranasal infection of mice with a virulent strain of influenza virus. RESULTS: All the obtained reassortants possessed 6 internal genes from strain-donor of attenuation and 2 genes, coding HA and NA-proteins from virulent strains. Ca reassortants were characterized by ts and ca phenotype, had antigenic specificity and good immunogenicity, had high protective effectiveness. CONCLUSION: The data obtained indicate on the perspectiveness of ca strain A/Krasnodar/101/35/59 (H2N2)as a donor of attenuation for live influenza vaccines.
Assuntos
Imunização , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Vacinas Atenuadas/imunologia , Animais , Embrião de Galinha , Temperatura Baixa , Humanos , Vírus da Influenza A Subtipo H2N2/imunologia , Vírus da Influenza A Subtipo H2N2/patogenicidade , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H3N2/patogenicidade , Vacinas contra Influenza/uso terapêutico , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Camundongos , Vacinas Atenuadas/uso terapêuticoRESUMO
Cold-adapted (CA) strains A/Krasnodar/35 and B/Victoria/63 were isolated using passages of A/Krasnodar/101/59 and B/Victoria/2/87 wild type strains at low temperatures. The resulting CA strains possessed TS and CA phenotypes and had a reduced ability to reproduce in mouse lungs and nasal turbinates. They displayed a high protective efficacy in experiments on mice. The two CA strains reproduced well in chick embryos and MDCK cell line without change of TS and CA markers. The CA A/Krasnodar/35 strain during passages at low temperature acquired 13 mutations in the 6 internal genes, 8 of those mutations led to amino acid changes. The CA B/Victoria/63 strain acquired 8 mutations in the internal genes, 6 of which led to amino acid changes. The intranasal vaccination of mice with the CA A/Krasnodar/35 strain led to a transitory suppression of various lymphocyte subpopulations, as well as to an increase in the number of some other cell types. The CA strains in question may be used in the future as attenuation donors for live influenza vaccines.
Assuntos
Adaptação Fisiológica/genética , Temperatura Baixa , Vírus da Influenza A Subtipo H2N2 , Vacinas contra Influenza , Mutação , Substituição de Aminoácidos , Animais , Linhagem Celular , Embrião de Galinha , Cães , Humanos , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/imunologia , Vírus da Influenza A Subtipo H2N2/metabolismo , Vacinas contra Influenza/biossíntese , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Camundongos , Vacinas Atenuadas/biossíntese , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologiaRESUMO
A ts+ ca- (non-temperature-sensitive, non-cold-adapted) revertant of the A/Leningrad/134/47/57 ca strain influenza virus [A/Leningrad/134/47/ts+18/1957(H2N2)], obtained in our previous study, lost phenotypic manifestation of ts mutations by the PB2, NP and NS genes, although, according to sequencing data, it acquired only two true reversions of a mutation in the PB2 and PB1 genes. Direct sequencing showed the appearance of 27 additional mutations (13 coding) in the genes encoding the PB2, PB1, PA, NP, M and NS proteins of the revertant, along with the above-mentioned two true reversions. We conjecture that some of these mutations suppressed phenotypic manifestation of ts mutations in the NS and NP genes.
Assuntos
Vírus da Influenza A Subtipo H2N2/fisiologia , Aclimatação , Temperatura Baixa , Teste de Complementação Genética , Genótipo , Humanos , Vírus da Influenza A Subtipo H2N2/genética , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Mutação , Fenótipo , Polimorfismo Conformacional de Fita Simples , Recombinação Genética , Proteínas não Estruturais Virais/genéticaRESUMO
A ts+ revertant of cold-adapted (ca) strain A/Leningrad/134/47/57--the attenuation donor for live influenza reassortant vaccines--was obtained by passages of the ca strain in chick embryos at nonpermissive temperatures. The ts+ revertant acquired the ability to grow in chick embryos at 40 degrees C and lost the capacity to reproduce there at 25 degrees C. A complementation-recombination test using the fowl plague virus (FPV0 ts-mutants showed the loss of the ts-phenotype in the RNA-segments of ts+ revertants' genome coding for PB2, NP, and NS (NS2) proteins. However, PCR-restriction analysis revealed a true reversion in RNA-segment coding for PB2 protein only. All the investigated mutations in the ts+ revertant genome were preserved. This phenomenon could be explained by the appearance of intragenic and extragenic suppression mutations in the ts+ revertant genome. The data of the complementation-recombination test suggest that reversion of ts-phenotype occurs more frequently due to extra- or intragenic suppression rather than as a result of a true mutation loss. Estimation of the genetic stability of vaccine ca strains of influenza virus should be based on the combined use of PCR-restriction and complementation tests.
