114 ALICK ISAACS 



There are many difficulties in applying these techniques directly to the 

 study of animal viruses, where, until recently, it has not been possible to 

 infect uniform populations of isolated suspended cells. The advantages 

 enjoyed by workers with bacteriophages are that the infectivity of their 

 virus is usually stable at 37°C; that adsorption of phages to their host cells is 

 usually efficient; that the assay of infectivity is simple and has a relatively 

 low error; that it is possible to assay without difficulty the number of infected 

 cells; that the bacterial cells can be effectively isolated from one another to 

 prevent spread of virus from cell to cell; and that in the cases which have been 

 studied most there is a one-to-one ratio between infectivity and total virus 

 particles present, as determined by electron microscopic counts (Luria et at., 

 1951). In some or all of these respects workers with animal viruses have been 

 at a great disadvantage. Most studies of this kind have been carried out with 

 whole animals, tissues such as the chick chorioallantoic membrane, or 

 populations of cells cultured in vitro under conditions where it is not known 

 how many cells are supporting virus multiplication. In addition, many animal 

 viruses are inactivated at 37°C. at a rate which appreciably affects the 

 interpretation of the results of virus absorption studies, and often the absorp- 

 tion itself is not very efficient. In titrations carried out by methods other than 

 pock- or plaque counting techniques the error of the titrations is usually high 

 (Dulbecco, 1955); finally, with all animal viruses which have been adequately 

 studied so far the minimal infective dose corresponds to at least 5 to 10 virus 

 particles counted by electron microscopy. These difficulties should make for 

 caution, and perhaps a little sympathy, in the interpretation of studies on the 

 eclipse phase of animal viruses. 



2. Stnall Animal Viruses 



Rubin et al. (1955) infected a monolayer of chick embryo cells with a high 

 multiplicity of western equine encephalitis virus. After allowing 30 minutes 

 at 37°C. for virus adsorption, the monolayer was washed and trypsinized and 

 the suspended cells washed and diluted greatly to prevent reinfection of cells. 

 This was taken as zero time and the diluted cells were then incubated at 37°C. 

 in buffer. At intervals, samples were removed and assayed for the total 

 number of infected cells by direct plating on a cell monolayer. At the same 

 time, the intracellular virus content was measured by plating out cells which 

 had been washed to remove superficially adsorbed virus and disintegrated by 

 ultrasonic vibration (a procedure which was shown to have no detectable 

 effect on virus infectivity). The sample at time "zero" was found to contain 

 1.3 X 10 4 infected cells per milliliter, but 2.9 ml. of an aliquot, when disinte- 

 grated, revealed no intracellular infective virus. This corresponds to a recovery 

 of less than 1 virus particle in about 40,000 infected cells. 



