Procedures which make possible the enumeration of both infective and hemagglutinating influenza A virus particles have been developed and used in a quantitative investigation on the reproduction of the agent. Infective particles were found to be highly unstable and their half-life was only 147 minutes in allantoic fluid at 35°C. both in vitro and in vivo. The instability of infective particles provides an explanation for the rapid accumulation of non-infective particles which retained the hemagglutinating property. The number of non-infective (N) particles was determined from the difference between the number of hemagglutinating (H) particles and the number of infective (I) particles as indicated by the relation: [N] = [H]– .
When the half-life of infective particles was taken into account, both infective and hemagglutinating particles were found to disappear from the allantoic fluid; i.e., were adsorbed by the allantoic membrane, at the same logarithmic rate after inoculation. Inoculation of any number of particles up to 3 x 107 was followed by a constant and progressive decrease in the proportion of unadsorbed particles from 0 to 4 hours. Approximately 20 per cent of particles were unadsorbed at 2 hours and about 5 per cent at 4 hours. Inoculation of 3 x 108 or more particles led to a larger proportion of unadsorbed particles at 4 hours. The maximum number of particles adsorbed was computed to be about 1.6 x 109.
The concentration of both infective and hemagglutinating particles increased rapidly in the allantoic fluid after 4 hours when any number of infective particles up to 3 x 107 was inoculated. With such inocula, the rate of increase during the logarithmic period was constant and the time to double the concentration of infective or hemagglutinating particles was 46 minutes. With larger inocula, i.e. 3 x 108 particles, the concentrations of infective and hemagglutinating particles did not increase until after 8 hours and the rate of increase was much slower. The time to double the concentration of either then became 92 minutes.
The number of infective particles was approximately equal to the number of hemagglutinating particles during the logarithmic increase period when any number of infective particles up to 3 x 106 was inoculated and no more than 106 non-infective particles were included in the inoculum. This finding was taken to indicate that all or almost all particles produced and released under these conditions were infective. That such particles became inactivated rapidly and led to the accumulation of an increasing number of non-infective particles after the logarithmic period can be explained by the short half-life of infective particles.
The number of infective particles was no larger than one-tenth the number of hemagglutinating particles during the logarithmic increase period after 3 x 107 or more infective particles had been inoculated or when smaller inocula were used which also contained 3 x 107 or more non-infective particles. Non-infective particles prepared in vitro at 35° or 22°C. were as effective as those which accumulated in vivo in diminishing the proportion of infective particles in the yield. The extent of the reduction in the proportion of infective particles was directly related to the number of non-infective particles included in the inoculum. The yield of hemagglutinating particles was diminished when the inoculum contained 3 x 107 or more non-infective particles. The rate of increase was reduced so that the time to double the concentration became 92 minutes when the inoculum contained 3 x 108 non-infective particles.
It appears from these findings that the single condition which will lead to the emergence of non-infective particles during the logarithmic period is a high initial particle-cell ratio. Because non-infective particles are equally as effective as infective particles in producing this result, it seems probable that the appearance of non-infective but hemagglutinating particles is not a necessary accompaniment of the reproductive process.