RESUMO
Live attenuated cold-adapted influenza vaccine (LIV) has been used in Russia for over 50 years and proved to be safe and effective. Currently, Russian reassortant LAIV is based on influenza AILeningrad/134/17/57 (H2N2) and B/USSR/60/69 Master Donor Viruses (MDVs) which are cold-adapted (ca), temperature-sensitive (ts), and attenuated (att), respectively. The MDVs are used to generate attenuated reassortant vaccine viruses containing the surface antigens of current wild type (wt) influenza A (HINI) and A (H3N2) viruses and wt influenza B virus. The ca/ts/att phenotype of these viruses limits replication in the upper respiratory tract. Reassortment typically yields numerous viruses with different genome constellations, rapid screening is needed to select proper vaccine viruses. In this study, screening of reassortant vaccine strains for live attenuated influenza vaccine generated from currently circulating influenza A and B viruses by RFLP assay is described.
Assuntos
Bioensaio , Enzimas de Restrição do DNA/metabolismo , Vacinas contra Influenza/genética , Polimorfismo de Fragmento de Restrição , Vírus Reordenados/genética , Vacinas Atenuadas/genética , Adaptação Fisiológica/genética , Animais , Embrião de Galinha , Temperatura Baixa , Primers do DNA/química , Primers do DNA/metabolismo , Genoma Viral , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/imunologia , Vírus da Influenza B/genética , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Pandemias , Fenótipo , Vírus Reordenados/imunologia , Federação Russa , Vacinas Atenuadas/imunologia , Replicação ViralRESUMO
The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6:2 reassortant viruses containing the surface antigens hemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, and conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6:2 reassortant viruses were selected from co-infections between master donor virus and wild type viruses that theoretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6:2 reassortant viruses are allowed as vaccine viruses, screening needs to be both rapid and unambiguous. The screening of the reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets was described to select both influenza A and influenza B 6:2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine.
Assuntos
Genoma Viral/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza B/genética , Vacinas contra Influenza/genética , Influenza Humana/prevenção & controle , Vírus Reordenados/genética , Animais , Embrião de Galinha , Coinfecção/virologia , Hemaglutininas/genética , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza B/imunologia , Influenza Humana/epidemiologia , Neuraminidase/genética , Pandemias , RNA/genética , RNA/isolamento & purificação , Vírus Reordenados/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologiaRESUMO
AIM: To identify the genes that are responsible for attenuation of donor viruses for live influenza vaccine. MATERIALS AND METHODS. Analysis of phenotypical properties of reassortants of wild type A and B influenza viruses with A/Leningrad/134/17/57 (H2N2) (A17) and B/USSR/60/69 (B60) master donor viruses was performed by comparison of their capability to grow at different temperatures in chicken eggs or/and MDCK cells. RESULTS: Ts phenotype of 178 reassortants of A17 with current non-ts influenza A wild type viruses and 33 reassortants of B60 with current non-ts influenza B wild type viruses were evaluated. Reassortants inherited two polymerase genes PB2 and PA or PB 1 from A17 regularly demonstrated ts phenotype. The polymerase PA and PB2 gene segments of B60 independently controlled manifestation of ts phenotype of B60 based reassortants. The other nonpolymerase genes played no role in manifestation of ts phenotype of reassortants A17 and B60 viruses. CONCLUSION: The molecular basis for the development ts phenotype of both A and B influenza vaccine reassortant viruses determined by polymerase genes complex.