Measuring the mutation rate during the growth of a single plaque. Plaque formation was performed in confluent monolayers of MDCK cells infected with A and B viruses. A well-isolated parent plaque of approximately 2 mm in diameter was picked at an appropriate time postinfection. To clone selected individual descendant viruses in the parent plaque, a second plaque formation was performed using the virus yield directly from the parent plaque. All plaque viruses were eluted from the agar plug into 300 μl of minimal essential medium containing 0.2% bovine serum albumin, made into aliquots, and stored in a deep freezer (−80°C) until used. The harvested viruses were used without further modification for sequential analysis of their NS genes, which reveals the sequence of the major virus in the plaque. The magnified views of the plaques show the viruses that exist within them. Star, viruses carrying NS gene sequences identical to that of the major virus in the parent plaque; triangle, diamond, spade, club, heart, and square, viruses carrying NS gene sequences different from that of the major virus in the parent plaque. The two triangles in the parent plaque represent a clone line. One of the descendant plaques is descended from a mutant of this type.

Comparison of the generation times of A and B viruses in MDCK cells. Confluent monolayers of MDCK cells (5 × 10⁵ cells) were infected with A/Aichi/1/87 and B/Aichi/29/99 at a multiplicity of infection of 8, kept at room temperature for 1 h, washed three times with minimal essential medium, and then incubated in 500 μl of overlay medium (Table 1, footnote a) excluding agar and DEAE-dextran, at 34 or 37°C. At 0 and 5 h postinfection and at each subsequent hour, a sample of the supernatant was removed and analyzed by plaque titration in MDCK cells. The PFU per 500 μl are shown versus the hours postinfection. (Inset) The generation times were determined as the point of maximum release of PFU per hour in each one-step growth experiment (1, 7). This is generally equivalent to the point at which the PFU reaches 50% of its final value, estimated here by averaging the total PFU at 11, 12, and 14 h. The width of the distribution about the mean was likewise estimated by noting the points at which the PFU reached 16 and 84% of its final value, corresponding to plus and minus 1 standard deviation (1). The change in temperature from 34 to 37°C, tested for A/1/87 only, appeared to have no significant effect on the mean generation time.