124 ALICK ISAACS 



cells in association with superficial virus and may later mix with intracellular 

 virus on grinding the cells. Gostling and Bedson (1956) found that chick 

 embryo cell suspensions incubated with herpes simplex virus overnight at 

 4°C. and then treated with diluted antiserum overnight at 4°C. contained 

 throughout the lag period about one-tenth the amount of virus of control 

 cells in which the antiserum treatment was omitted. In these experiments 

 evidence was shown that there was insufficient antiserum present to affect the 

 results of the infectivity titrations and the findings are attributed to neutraliza- 

 tion of superficially adsorbed extracellular virus. As mentioned earlier, the 

 level of virus extractable from the cells then declined to one-tenth of the 

 initial level, but this fraction of the initially absorbed virus was not neutralized 

 by treatment with antiserum in their experiments. In the experiments of 

 Yoshino and Taniguchi (1956) an antiserum in a 1/100 dilution was used to 

 treat chorioallantoic membranes one-half hour after inoculating herpes 

 simplex virus by the cover slip technique mentioned above. The use of anti- 

 serum was combined with an elaborate wash-drying procedure involving 

 numerous washings for each membrane in buffered saline with careful 

 drying on filter paper between washes. With this method about 0.0015 to 

 0.004 % of the inoculum was recoverable during the lag period compared 

 with about a 20 % recovery in membranes not treated in this way. These 

 workers also attempted to remove superficially adsorbed virus by experiments 

 involving washing the membrane in situ, followed by sponging with gauze 

 after each wash. After 5 washings with sponging between each, the amount of 

 recoverable virus was about 1 % of that present before washing; this degree 

 of washing apparently did not harm the cells too much, as judged by the 

 yield of virus from similar membranes incubated at 35°C. However, with 6 

 washings, involving sponging with 3 changes of gauze between each washing, 

 the yield of virus was drastically reduced following incubation at 35°C. Such a 

 traumatic procedure might easily damage cells so that intracellular virus could 

 be washed away . These experiments display novel methods of trying to answer 

 this problem and it would be interesting to see them applied to vaccinia virus. 



That tissues which had adsorbed influenza virus when repeatedly washed 

 (15 times) in buffer continued to release virus into the washings was demon- 

 strated by Ackermann et al. (1955), and similar findings were noted after 12 

 washings of cells infected with herpes simplex virus (Gostling and Bedson, 

 1956). One method used to remove superficially adsorbed influenza virus was 

 to treat the intact chorioallantoic membranes with large doses of RDE, 

 followed by thorough washing before grinding (Isaacs and Edney, 1950). 

 With the use of RDE the recovery of infective virus was reduced 10-fold, i.e., 

 0.04 % of the seed compared with 0.6 % in membranes which had not been 

 washed with RDE. Insufficient RDE was present to interfere with the assay 

 of infectivity. 



