RESUMO
Live, cold-adapted, temperature-sensitive (ca/ts) Russian influenza A vaccines are prepared in eggs by a 6:2 gene reassortment of the ca/ts donor strain A/Leningrad/134/17/57 (H2N2) (Len/17) with a current wild-type (wt) influenza A strain contributing hemagglutinin (HA) and neuraminidase (NA) genes. However, egg-derived reassortant vaccines are potentially more problematic to manufacture in large quantities than vaccines from cell-based procedures. To compare egg- and cell culture-derived reassortant vaccines, we prepared in Madin Darby canine kidney (MDCK) cells two cloned, ca/ts reassortants (25M/1, 39E/2) derived from Len/17 and a wt reference strain A/New Caledonia/20/99 (H1N1) (NC/wt). Both 25M/1 and 39E/2 reassortants preserved the ca/ts phenotype and mutations described for internal genes of the A/Len/17 parent. When compared to a commercial, egg-derived ca/ts Russian A/17/NC/99/145 (H1N1) New Caledonia vaccine (NC/145), the MDCK-derived reassortant 39E/2 vaccine conferred similar levels of protection in ferrets challenged i.n. with 7 x 10(10) pfu of NC/wt. In a dose-ranging study, the protective vaccine dose for 50% of ferrets (PD50) was less than 1.2 x 10(4) pfu for the 25M/1 vaccine derived by recombination and amplification in MDCK cells. Clonal isolates of ca/ts influenza A/New Caledonia/20/99 (H1N1) obtained by recombination and amplification entirely in MDCK cells can be highly protective i.n. vaccines.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Administração Intranasal , Animais , Líquido da Lavagem Broncoalveolar/virologia , Linhagem Celular , Embrião de Galinha , Modelos Animais de Doenças , Cães , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vacinas contra Influenza/administração & dosagem , Líquido da Lavagem Nasal/virologia , Neuraminidase/genética , Infecções por Orthomyxoviridae/imunologia , Vírus Reordenados/crescimento & desenvolvimento , Vacinação , Ensaio de Placa Viral , Proteínas Virais/genéticaRESUMO
A T4 RNA ligase based strategy is demonstrated that allows for the full characterization of 3' and 5' UTR regions of negative strand RNA viruses. Negative strand RNA viruses such as influenza have 3'OH and 5'P terminal ends that are capable of being ligated using T4 RNA ligase. Each segment can form a mixture of linear concatamers between like and different viral segments or can itself form a circular structure upon ligation. RT-PCR can then be performed on these circular RNA segments using gene specific primers subsequently allowing for the characterization of the true terminal sequence for each viral segment. The UTR regions of a number of influenza virus strains were defined accurately using this approach.
