Preparation and characterisation of attenuated cold-adapted influenza A reassortants derived from the A/Leningrad/134/17/57 donor strain.

The development of a rapid cell culture method for the preparation of cold-adapted (ca) influenza A reassortant viruses is described and compared with a currently used egg method. Mixtures of the ca donor A/Leningrad/134/17/57-ca (A/Len/17) and A/Beijing/32/92 (A/Beij/32), a recent H3N2 epidemic strain, were used to co-infect chicken embryo kidney (CEK) cell cultures; reassortant progeny were selected using an infectious centre assay. The assay was capable of detecting interference where the infectivity ratio for A/Len/17 and A/Beij/32 was 1:7. Progeny viruses were characterised genetically by amplification of defined regions within each of the six internal genes by PCR and identification of the products by restriction enzyme analysis. Reassortants were also tested for ca and temperature-sensitive (ts) phenotype and the identity of the surface antigens. The infectious centre assay was shown to be an effective method for isolating reassortant progeny that possessed the haemagglutinin and neuraminidase surface antigens of A/Beij/32. Reassortants with the six internal genes derived from the donor strain and possessing the ca and ts phenotype were readily obtained when (a) an infectivity ratio of 1:49 was used and (b) two plaque-to-plaque isolations of progeny virus were made after growth at 25 degrees C and one at 34 degrees C, both in the presence of antiserum to the donor strain.