Influenza virus vaccine candidate strains with greater yield potential than the naturally occurring wild type strains of influenza A virus have been produced by reassorting the wild type strain with a strain of influenza A/Puerto Rico/8/34 (PR8) with the ability to confer the """"""""high yield"""""""" phenotype. The hemagglutinin (HA) and neuraminidase (NA) of a wild type strain are selected by suppressing the progeny virions bearing the HA and NA of PR8 by incubation in the presence of an antiserum produced against PR8. With this strategy, the possibility exists that a minor component of the resulting virions may still include the genotype or phenotype of PR8 HA or NA. In order to develop a sensitive method for identifying whether the PR8 genome may be persistent, the polymerase chain reaction (PCR) technique has been adapted for identification of HA and NA genes from H1N1 and H3N2 viruses. The specificity of the primers chosen for DNA polymerization was first confirmed for both HA's and NA's using a battery of H1N1 viruses and H3N2 viruses. Subsequently, H1N1 and H3N2 viruses were mixed in varying ratio in order to determine the limit of detection of the method for NA. With a single session of 30 cycles of polymerization, it was possible to detect 1 N2 virion in 1000 N1 virions. With an additional session of 30 cycles using either the same set of primers or a set of primers for a sequence contained within the product from the first set of primers, it was possible to increase the sensitivity by 10 fold, although not consistently. Thus, by PCR it was possible to specifically identify a minor component at ratios of 1:1000- 1:10000. In marked contrast, the standard method of identification of neuraminidase by inhibition of enzymatic activity with a """"""""specific"""""""" antibody, was not consistently capable of identifying mixtures of N1 and N2 virions even at ratios of 1 part in 10. The results indicate that PCR is a more sensitive method of detection of genetically mixed influenza virus populations, and that PCR techniques can be very useful in a program to produce reassortant viruses.
