Sequential passage of influenza virus in embryonated eggs or tissue culture: Emergence of mutants

The emergence of influenza virus mutants was examined by passage of A/WSN/33 virus in embryonated eggs, in MDBK cells, and in MDCK cells. From a plaque-purified A/WSN/33 virus preparation, four independent clones were derived by 12 plaque-to-plaque passages in MDCK cells. Four clones were isolated following 8-12 plaque-to-plaque passages of A/WSN/33 virus in MDBK cells, and one clone was derived by 12 passages in embryonated eggs at limiting dilution. All nine clones independently derived by these passages showed differences in their oligonucleotide maps when compared to the map of the original preparation of plaque-purified A/WSN/33 virus. In contrast, 12 passages in embryonated eggs at low dilutions (10'-10' PFU per inoculum) resulted in an A/WSN/33 virus population which was indistinguishable by oligonucleotide mapping from the original preparation. The frequent emergence of influenza A virus variants upon plaque-to-plaque passage contrasts with the finding that three out of four vesicular stomatitis virus clones, which were derived from independent plaque-to-plaque passage in MDCK cells, were identical to the original preparation when compared by oligonucleotide mapping.