Deviation from the random distribution pattern of influenza A virus gene segments in reassortants produced under non-selective conditions

High-frequency reassortment of gene segments is characteristic for influenza viruses, and it is considered to be of significance for the origin of pandemic influenza. In order to analyze whether the segregation of genes in the reassortants is random, or it deviates from the random pattern, we inoculated embryonated chicken eggs simultaneously with two influenza viruses, A/WSN/33 (H1N1) and A/Duck/ Czechoslovakia/56 (H4N6), at a high multiplicity of infection. The virus yield was used for plaque cloning, and the genetic content of plaque isolates was determined by analysis of the mobility of virus-induced proteins in polyacrylamide gel (for NP and NS genes), partial sequencing (for M gene) and polymerase chain reaction analysis with strain-specific primers for the other genes. Out of 37 isolates, 27 were reassortants. The majority of the reassortants contained the HA gene of A/WSN/33 (H1N1) virus and the NP gene of A/Duck/Czechoslovakia/56 (H4N6) virus. The data demonstrate the previously unrecognized phenomenon of segment-specific deviation from the random distribution of parent genes in the reassortants. The results are discussed in connection with the problem of differential competition between influenza A virus gene segments in mixed infection and random versus non-random reassortment of gene segments under non-selective conditions.