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Temperature-sensitive (ts ) reassortant vaccine strains derived from the A/Udorn/72ts -1A2 donor virus were not sufficiently stable genetically in humans. We therefore sought to produce a new, more stable donor virus. We had previously identified a stablets virus with ats P3 gene and in the current study identified another relatively stable single-lesionts virus with ats mutation in the NP gene. A newts reassortant virus was constructed by mating these two single mutants and by isolating three reassortant progeny, clones 20, 53, and 55, that contained both ats P3 and ats NP gene. These reassortant progeny possessed a 37 to 38°C shutoff temperature and were as restricted in their replication in hamster lungs as the A/Udorn/72ts -1A2 virus. All isolates from the lungs and nasal turbinates of hamsters were temperature sensitive. An in vitro stress test was used to determine whether the newts P3ts NP reassortant virus would undergo loss of itsts phenotype after replication at semipermissive temperature. Clone 20 and 55 reassortants underwent progressive loss of theirts phenotype in vitro, although at a rate slightly less than that of the A/Udorn/72ts -1A2 virus. The level of genetic stability after replication in vivo was assessed in cyclophosphamide-treated hamsters in which virus replication continued for up to 15 days. Again, both the A/Udorn/72ts -1A2 and the newts P3ts NP reassortant clone 55 manifested a progressive loss of temperature sensitivity after 7 days of replication. Clone 55 virus lost temperature sensitivity significantly less rapidly than the A/Udorn/72ts -1A2 virus. These results indicated that, although the newts P3ts NP reassortant virus was more stable than the A/Udorn/72ts -1A2 virus, it nevertheless underwent progressive loss of temperature sensitivity after replication in vitro and in vivo. Therefore, it does not appear to be a satisfactory donor virus. This experience plus that gained earlier with otherts mutants of influenza A virus suggest that influenza A virus mutants that rely solely upon theirts phenotype for attenuation are unlikely to exhibit the phenotypic stability required of a vaccine virus. Other genetic techniques are needed to produce more stable influenza A virus strains.

Production and Level of Genetic Stability of an Influenza A Virus Temperature-Sensitive Mutant Containing Two Genes with ts Mutations