82 F. B. BANG 



with measurements of the infectious virus produced. By infecting at a given 

 time with an amount of virus sufficiently large to infect most of the cells, it 

 was possible to follow sequentially the cellular changes. They found the same 

 groups of particles with dense centers as described by Morgan et al. (1954a) 

 but reported that they appeared in the cytoplasm and the nucleus at the 

 same time. They classified them as (a) particles with a single membrane 

 which ranged in size from 60 to 100 fi; (b) particles with two membranes 

 (120 to 180 fx), which occurred both in the cytoplasm and the nucleus; and 

 (c) particles about 180 ju, in diameter which had a double central body. 

 These last were found both in the cytoplasm and on the external surfaces 

 of the cell. Some of the particles appeared to have two nuclei, each 

 with its own membrane, the pair juxtaposed within a single outer 

 membrane. 



Although mitochondrial changes produced by herpes simplex have been 

 briefly described in electron microscope studies of herpes-infected tissue 

 (Bang, 1955), they have not been studied in detail, and knowledge concerning 

 the effect of this virus on the cytoplasm is lacking. A study of differentially 

 stained "inclusion bodies in experimental herpetic infections of rabbits" by 

 Cowdry and Nicholson (1923) led to inconclusive results on this point. Yet 

 this might be particularly important material, since the herpesvirus 

 apparently travels long distances along the axon of a nerve cell as it 

 progresses from cell to cell and leaves behind the characteristic nuclear 

 lesion. 



E. Fluorescent Antibody Staining 



A study with fluorescent antibody by the double antibody technique on 

 two strains of herpesvirus (one recently isolated and one "classical strain" in 

 unfixed preparations of trypsinized tissue cultures of human amnion and 

 infant mouse kidney cells) showed a specific peripheral fluorescence in these 

 cells. Despite the demonstration of morphological changes at 18 hours, of 

 intranuclear lesions at 24 hours, and cell degeneration at 31 hours, the 

 fluorescent staining of the nucleus itself was not clear in any of these cultures 

 (O'Dea and Dineen, 1957). This limited staining of the cell may, however, as 

 the authors point out, have been due to "impermeability of unfixed cells" 

 to the globulin. These results are contrasted to Lebrun (1956), who found 

 virus antigen first as small foci within the nuclei of infected carcinoma cells 

 in tissue culture. Later large fluorescent masses accumulated in the nucleus, 

 followed finally by cytoplasmic accumulation of antigen. It was at this last 

 stage that the classic intranuclear inclusion was apparent. Lebrun refers to 

 this as an intranuclear scar. Large amounts of virus were used and thus, 

 presumably, most of the cells were infected early. 



