Stability of the mutant genes of the cold-adapted influenza virus donor strain. The genetic stability of the PB2 gene of the cold-adapted (ca) donor virus, used to prepare reassortant vaccine strains, was studied by subjecting a single gene (PB2) reassortant virus to serial passage in cell culture at a non-permissive temperature. Genetic and sequence analysis of ts+ """"""""revertant"""""""" viruses indicated that the temperature-sensitive (ts) and attenuation phenotypes specified by the PB2 gene of the ca influenza A donor virus were suppressed by a single amino acid substitution in the PA gene that codes for another protein of the polymerase complex. Construction of an attenuated influenza A virus by introduction of specific mutations into the cDNA of an influenza A virus gene and rescue of the mutant gene into a viable chimeric virus. A chimeric neuraminidase (NA) gene that contained the coding region of the influenza A/WSN/33 NA and the 3' and 5' nontranslated sequences of the nonstructural (NS) gene of the influenza B/Lee virus was introduced into the genome of influenza A/WSN/33 virus by transfection, gene rescue and reassortment. The resulting reassortant virus was highly attenuated for mice and replicated poorly in the upper respiratory tract Nonetheless, prior infection with the mutant protected mice from virulent influenza A/WSN/33 virus challenge. The virus bearing the chimeric gene, therefore, has many properties that make it desirable for use as a live attenuated vaccine virus and thus it represents the first candidate live attenuated influenza A virus vaccine generated by recombinant DNA technology. Intragenic suppression of the ts phenotype specified by a 12 amino acid deletion in the NS gene. The ts phenotype specified by a 12 amino acid (AA) deletion (AA 66-77) in the NS1 protein of influenza A virus was suppressed by a single amino acid substitution of valine for alanine at AA23. The observation that a point mutation can alter the phenotype specified by an appreciable deletion mutation indicates that use of deletion mutation to achieve attenuation does not insure the genetic stability of the resulting virus mutant in vivo.
