BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 119 



declined between the third and sixth hours to about 10 % of the level 

 initially present. Gostling (1956) later showed that no further virus was 

 recovered from such cells by extracting the nuclei with 6 % sodium chloride 

 solution. Yoshino and Taniguchi (1956) have investigated in detail the 

 question of an eclipse phase for herpes simplex virus. The virus was applied to 

 a glass cover slip, which was then inverted onto the dropped chorioallantoic 

 membrane. Under these conditions and over quite a large range in inoculum 

 size, about 80 % of the inoculum was apparently absorbed by the cells 

 within 30 minutes and 20 % remained unabsorbed for some hours. When 

 extracts of chorioallantoic membrane were prej)ared, the residual unabsorbed 

 virus became included with the virus present in the membrane, so it was 

 necessary to carry out elaborate washing of the membrane to remove super- 

 ficially absorbed virus. These experiments are discussed in Section C, which 

 deals with the significance of the virus recovered during the lag period. 



Recently Stoker and Ross (1958) studied the growth of herpes simplex 

 virus in sheets of HeLa cells. Infection was initiated at high multiplicity of 

 exposure and most of the extracellular virus was then removed by washing 

 and treatment with antiserum. The proportion of intact infected cells was 

 determined by titrating cell suspensions on the chick chorioallantoic mem- 

 brane during the lag period; in different experiments it varied between 2 and 

 88 % of the total number of cells. After disintegration in distilled water hi a 

 microblendor, cell debris gave a significantly lower yield of infective virus 

 than intact cells, the recovery measured in this way being about 10 %. 

 (Neither the method of disintegration nor the cell debris reduced the 

 infectivity of free virus.) Thus the amount of virus detected in the lag period 

 corresponded to about one-tenth of the number of intact infected cells found 

 to be present. Therefore, the majority of cells which would ultimately yield 

 virus did not reveal any infective virus when disintegrated during the lag period. 



The very large "viruses," or Chlamydozoaceae, have some interest in 

 connection with the question of an eclipse phase, since they occupy a position 

 intermediate between smaller viruses and rickettsiae. Girardi et al. (1952) 

 inoculated chick embryos by the allantoic route with about 2 x 10 6 infective 

 doses of meningopneumonitis virus and measured the content of virus in 

 extracts of chorioallantoic membrane after different intervals of incubation. 

 There was an increase in titer in the membranes as virus was taken up and 

 this was followed by a slow and constant decline over a period of 20 hours. At 

 that time the titer in the membranes was about 1 % of that initially present. 

 Morgan (1956) has studied the growth of psittacosis virus in chick embrvo 

 mince which had been starved of essential metabolites. After inoculating 

 10 3 - 5 infective doses (LD 50 ) of virus, the infectivity was rapidly lost and after 

 6 hours' incubation in starved cells titration of ground cells did not reveal any 

 virus. In this case the recovery of virus was less than 0.03 % of that inoculated. 



