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 o f live attenuated vaccine reassortants. The approach is based on PCR a mplification of approximately 150-300-nucleotide-long regions of indiv idual RNA segments that include the unique nucleotide positions, follo wed by restriction nuclease treatment of the DNAs obtained with specif ic restriction endonucleases. Restriction sites recognized by chosen n ucleases either existed or were created during PCR in the genome of on e (but not the other) parent strain. The technique requires a minimal amount of infectious virus (approx. 100 mu l of allantoic or tissue cu lture 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 segm ent or the presence of a mutation. The method is beneficial for genome composition analysis of reassortant vaccine strains as well as for in vestigation of the genetic stability of live attenuated vaccines durin g replication in vaccinees.