BIOLOGICAL ASPECTS OF INTRACELLULAR STAGES OF VIRUS GROWTH 121 



Hoyle (1948) described a typical experiment in which an egg was inoculated 

 with 1024 nonagglutinating units of virus. After 3 hours' incubation, only 

 about 10 % of the hemagglutinin remained in the allantoic fluid and pre- 

 sumably the rest had been taken up by the cells. However, when the membrane 

 was ground with sand and incubated for 8 hours to allow elution of virus 

 from cells, no hemagglutinin could be detected; this is less than 0.5 % of the 

 amount that might have been expected to be present. Similar findings are 

 recorded by Henle and Henle (1949), i.e., no hemagglutinins were recoverable 

 from membranes in the first 2-3 hours after the use of very large inocula. But 

 in this study no attempt was made to remove normal inhibitors of agglutina- 

 tion in the membrane; this factor could, in theory, influence the recovery of 

 hemagglutinin from membrane extracts, depending on the dosage and strain 

 of virus used. However, Isaacs and Edney (1950) showed that the use of RDE 

 (receptor-destroying enzyme of Vibrio cholerae) to inactivate chorioallantoic 

 membrane inhibitor did not increase the recovery of hemagglutinin. In their 

 experiments, less than 1 % of the hemagglutinin taken up by the cells 

 could be recovered in membranes which were ground and treated with RDE. 

 They also tested membranes which had absorbed large doses of influenza virus 

 for evidence of virus enzymatic (neuraminidase) activity during the lag period. 

 Chorioallantoic membranes which had absorbed between 400 and 700 agglu- 

 tinating doses of virus, each, were incubated for short periods at 37°C. in 

 saline to allow elution of superficially absorbed virus. They were then ground 

 and aliquots were incubated at 37 and 0°C. for 18 hours and the agglutinating 

 inhibitory titer of the membranes measured. If any virus enzyme was present 

 it should inactivate the inhibitor of agglutination during incubation at 37°C. 

 but not at 0°C. In fact, no such enzymatic activity was demonstrated, 

 whereas control membranes incubated with 100 agglutinating doses added 

 after grinding had their agglutinating inhibitory titer reduced by more than 

 96 % under the same conditions. The fact that during the lag period neither 

 the hemagglutinating nor enzymatic activities of the virus could be detected 

 suggested that a fundamental change in the majority of the virus particles 

 occurred after they entered the cells. Confirmation of this idea was obtained 

 in similar experiments with heat-inactivated virus. Very large doses of heated 

 virus (roughly 5000-10,000 agglutinating doses of virus per egg) were absorbed 

 by chorioallantoic membranes, which were then ground and treated with 

 RDE. Such extracts showed no evidence of viral hemagglutinin, or of 

 antigenic activity as determined in vitro by antibody-combining activity, 

 or in vivo by antigenicity in mice. By contrast, heated virus incubated with 

 chorioallantoic membrane extract in vitro, and then treated with RDE, 

 showed high activity by all three tests. 



Tamm and Tyrrell (1954) studied the recovery of influenza virus hemag- 

 glutinin after 1 hour's incubation with pieces of chorioallantoic membrane 



