134 ALICK ISAACS 



from the cells within about half an hour. Again the rapid liberation may have 

 been a reflection of the culture conditions. 



Howes and Melnick (1957) have also studied the growth of type 1 polio virus 

 in monkey kidney cell monolayers and they ensured that a single cycle of 

 growth was observed by infecting their cells at a multiplicity of 4 or more. 

 They found that after a lag period of 3 hours there was a rapid and, at first, 

 exponential increase in the amount of cell-associated virus with the produc- 

 tion of approximately 100 plaque-forming units of virus per infected cell. 

 At about 5i hours, after the initiation of infection, 50 % of the total virus 

 yield had been produced. The yield of extracellular virus lagged behind the 

 cell-associated virus by about 1 hour. 



B. Western Equine Encephalitis Virus 



Dulbecco and Vogt (1954) used a dilution technique to make one-step 

 growth curves of western equine encephalitis virus grown in chick embryo 

 cell suspensions. The lag period was about 2i hours with a multiplicity of 4, 

 and 3^ hours with a multiplicity of 0.15. Once virus release commenced there 

 was an exponential rise in the titer of extracellular virus for about 1| hours, 

 followed by a slowing, with the maximal yield at 6-8 hours. The yield of 

 virus was approximately 100-200 infective particles per cell, but conditions 

 of cultivation were probably not optimal, since a yield of 200-1000 infective 

 particles per cell was found when the virus was grown in a cell monolayer. 

 With a high multiplicity of infection the yield of virus per cell was consis- 

 tently higher than at low multiplicity, suggesting that more than one virus 

 particle per cell can take part in the growth process. This last finding is an 

 extremely interesting one, and should be tested in other virus systems with 

 simultaneous study of the intracellular (or cell-associated) virus and the 

 extracellular virus. 



Rubin et al. (1955) studied the growth of this virus in a similar system but 

 with particular attention to the rate at which intracellular or cell-associated 

 virus developed. Monolayers were infected at high multiplicity and after a 

 30-minute absorption period the cells were washed and trypsinized and the 

 suspended cells diluted greatly. After a lag period of about 1^ hours the cell- 

 associated virus, measured after rupturing the cells by ultrasonic vibration, 

 was found to increase exponentially for a period of nearly 3 hours, the virus 

 doubling in amount every 15 minutes. As described earlier, no virus could be 

 detected in these cells at zero time, i.e., the time at which the cell dilution was 

 made. Rubin et al. calculated that the average maximum number of cell- 

 associated infective virus particles per cell at the end of the period of exponen- 

 tial rise was only 4 to 10, although each cell had spontaneously released 100 

 infective particles, i.e., the virus is very rapidly released when formed (this 



