PCR restriction analysis of genome composition and stability of cold-adapted reassortant live influenza vaccines.

Using cold-adapted master donor strains of influenza virus as a model, an approach was developed that exploits unique nucleotide differences between the donor strains and wild-type influenza viruses for rapidly and simply determining the genome composition and genetic stability of live attenuated vaccine reassortants. The approach is based on PCR amplification of approximately 150-300-nucleotide-long regions of individual RNA segments that include the unique nucleotide positions, followed by restriction nuclease treatment of the DNAs obtained with specific restriction endonucleases. Restriction sites recognized by chosen nucleases either existed or were created during PCR in the genome of one (but not the other) parent strain. The technique requires a minimal amount of infectious virus (approx. 100 microliters of allantoic or tissue culture fluid with a haemagglutination titre 1:4-1:8 or less) and allows rapid (within about 10 h) determination of the origin of the RNA segment or the presence of a mutation. The method is beneficial for genome composition analysis of reassortant vaccine strains as well as for investigation of the genetic stability of live attenuated vaccines during replication in vaccinees.