METCALF 



tinued to fall throughout the 24 hour test period. Sometime after 12 

 hours the production of enzyme withinthe membranes was detected, 

 with a steady increase shownduringthe remainder of the test period. 

 Confirmation of the fabrication of enzyme within the infected allan- 

 toic membrane was obtained in separate experiments by detection of 

 a rise of free neuraminic acid as measured by the TBA assay. The 

 level of free neuraminic acid in infected membranes began to in- 

 crease at 12 hours after infection and continued to increase through 

 24 hours. No increase could be shown for the non- infected mem- 

 branes (Fig, 4). 



When experiments of this kind were conducted withA2 strains in 

 chick embryos incubated at 20° C, the following results were found. 

 There was no significant increase in the virus concentration of mem- 

 branes, no decrease in inhibitor level, and no enzyme formation was 

 detected. Again there was agreement between inhibitor reduction 

 titrations and TBA assays (Fig. 5). 



The findings obtained in chick embryos with A2 strains possessing 

 a high enzyme activity were next compared to the results found with 

 a PR-8 strain of low enzyme activity. Virus multiplication and en- 

 zyme production occurred, and the inhibitor level decreased accord- 

 ingly when the experiment was conducted at 37*^ C. At 20 C no virus 

 growth or enzyme production was detected. The inhibitor level 

 fluctuated but did not show a steady decline (Fig. 6). 



Throughout the study a virus inoculum of 1000 LD50 doses was 

 used. It was decided to determine the effect of increasing the doses 

 given in the inoculum upon the relationships of enzyme production 

 and inhibitor content. Accordingly, an inoculum of 10 ' LD^q doses 

 per embryo was used. The net result was an accelerated appearance 

 of enzyme and earlier decline of inhibitor. The virus content of 

 membrane reached an early peak accompanied by a rapid rise in en- 

 zyme production between 6 and 9 hours. Membrane inhibitor fell 

 rapidly between 2 and 6 hours (Fig, 7), 



The pattern of events emerging from these experiments confirmed 

 the existence of a direct relationship between virus multiplication 

 and the appearance of enzyme activity in the allontoic membrane. 



354 



