INACTIVATION OF VIRUSES 399 



maximum of aljoiit 50 A towards higher wavelengths iii RNA (but not in 

 DNA) after treatment with 0-33 to 0-67 M formaldehyde. The phenomenon 

 was interpreted as evidence of formation of Schiff's bases, involving the 

 amino groups of purines and pyrimidines. The reaction appeared to be 

 reversible. In addition to amino groups, hydroxy groups of ribose and 

 secondary acid groups of phosphoric acid would seem to provide theoretically 

 possible reactive sites. 



As a chemical widely used for j^roduction of "killed" virus vaccines, for- 

 maldehyde is of considerable practical importance; for that reason formol 

 inactivation of a number of viruses has been extensively studied. 



1. Tobacco Mosaic Virus 



Stanley (1936) and Ross and Stanley (1938), studying the effect of for- 

 maldehyde on TMV, fomid that the infectivity decreased in a regular fashion 

 in the course of treatment. They observed that inactivation during the first 

 12-18 hours proceeded approximately as a first-order reaction, but that more 

 extensive inactivation required much longer periods of treatment than 

 would have been expected on the basis of such an assumption. Lauffer and 

 associates (Fischer and Lauffer, 1949a,b ; Cartwright et al., 1956), quoting 

 Ross and Stanley, state the reaction to be of the first order. Actually this 

 statement implies only that the data were better fitted by an equation of 

 order 1 than by equations of orders or 2; it does not exclude the possibility 

 that an order of 1.1 would have fitted even better, which, indeed, is the case 

 in Ross and Stanley's experiments. 



However, on the assumption of a first-order specific reaction rate, constants 

 were calculated and the effect of external factors on the inactivation rate 

 determined. Formol-treated virus had a lower electrophoretic mobility, in- 

 dicating a loss of charge, no doubt mainly by coverage of amino groups. The 

 rate of change in mobihty was much lower than that of inactivation, indicat- 

 ing either that the two phenomena were not referable to the same chemical 

 reaction or else that infectivity was maintained only as long as all "essential" 

 groups remained intact, i.e. an irreversible change in one of a number of 

 identical groups would mean complete loss of activity but only a small change 

 in the net charge. As it was later shown that the inactivation rate was largely 

 independent of pH in the range of 4 to 8.5, the amino groups, as well as 

 several other reactive sites, could probably be considered of little significance 

 in inactivation; the most likely essential sites would have to be looked for 

 among the beta hydroxy groups of threonine and serine, the hydroxy groups 

 of ribose, and the nitrogen of the purine and pyrimidine bases of the nucleic 

 acid. Preinactivation oxidation of sulf hydryl groups with iodine, or coverage 

 of amino groups by acetylation or coupling with diazosulfanilic acid had no 



VOL. 1—27 



