BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 117 



described for small and medium -sized viruses. Briody and Stannard (1951) 

 and Crawford and Sanders (1952), working with vaccinia virus grown, 

 respectively, on the chick chorion and in the rabbit skin in vitro, described a 

 decrease in the number of infective particles that could be detected in the 

 lag period, but the uptake of virus by the cells was not measured, so that the 

 actual extent of the decrease is not known. Anderson (1954) inoculated eggs 

 with 250 infective doses of vaccinia virus on the chorion and measured the 

 amount of virus that could be recovered from ground membranes after 

 various periods of incubation. The number of infective doses recoverable at 

 30 minutes was 70; at 1 hour, 350; this gradually declined to 18 infective doses 

 at 9 hours, followed by a steep increase. This suggests that the virus was 

 gradually adsorbed to the membrane over the first hour and that, as the 

 virus entered the cells, there was a decrease in the amount of recoverable 

 virus to about 7 % of that inoculated; from the titers obtained at 1 and 2 

 hours, it appears that most of the virus inoculated was taken up by the cells. 

 Metcalf (1955) described a slightly greater decline in titer following inocula- 

 tion of 7 X 10 4 infective doses, but in this experiment the uptake of virus was 

 not measured. By contrast, others who have worked with vaccinia virus 

 report much higher recoveries during the lag period. The interpretation of 

 Maitland and Tobin's (1956) results is complicated, however, by what they 

 call the "enhancement effect." They found that when a vaccinia elementary 

 body suspension was prepared from rabbit skin by differential centrifugation 

 and inoculated on the chorion, the amount of virus recoverable from the 

 liquid on top of the membrane immediately after inoculation was considerably 

 higher than the apparent titer of the inoculum (5 to 22 times in some 

 experiments). This is presumably some sort of disaggregation effect; after this 

 apparent rise in titer the virus entered the membrane. Thereafter the total 

 virus concentration decreased, until at 2 to 4 hours it was 20 to 80 % of the 

 peak titer (or 2 to 4 times the amount apparently inoculated) and most of 

 this virus was recoverable from the membrane itself. Maitland and Ma.grath 

 (1957) studied this decline in titer in more detail, using the same virus grown 

 in chorioallantoic membrane in vitro. When pieces of chorioallantoic membrane 

 were incubated with virus for 10 minutes, about 20 to 40 % of the inoculum 

 became attached to the membrane. However, about three-quarters of this 

 virus could be removed by washing, and the remainder constituted the 

 baseline. Following incubation of washed pieces of membrane there was a slow 

 decrease in the amount of virus recoverable. At 8 to 12 hours, the minimal 

 titer was reached when about 20 to 50 % of the baseline value was recoverable. 

 A similar result was obtained with minced chick embryo cells. Trypsinized 

 cells were left in contact with vaccinia virus for 10 minutes at 37°C, 

 incubated in vitro, and titrated at intervals along with their suspending 

 medium after disintegrating the cells mechanically. The base-line titration 



