DEMONSTRATION OF THE SEQUENTIAL DEVELOPMENT OF VACCINIAL ANTIGENS AND VIRUS IN INFECTED CELLS: OBSERVATIONS WITH CYTOCHEMICAL AND DIFFERENTIAL FLUORESCENT PROCEDURES

Virus-induced alterations in vaccinia virus-infected HeLa cells have been followed by immunofluorescent and cytochemical techniques. In a time sequence study, infected cells show an early increase in cytoplasmic RNA content, followed by appearance of centers of viral DNA synthesis in the cytoplasm. The centers of synthesis were detected at 4 hours post infection, with the acridine orange fluorochrome stain as compared to 6 hours with Feulgen and methyl green-pyronin stains. Marginal fragmentation of the inclusion bodies was seen at 8 to 10 hours post infection, and appears to coincide with the first increase in cell-associated virus. With the immunofluorescent technique, it was found that the LS antigen of the virus can be detected at about 4 hours post infection. This is followed at 5 to 6 hours post infection by the appearance of the NP antigen. Both antigens are found only in the cytoplasm, and precede the appearance of the infective particle. The HA antigen, a by-product of virus-cell interaction, is not seen until about 10 hours post infection; that is, several hours after the appearance of both the LS and NP antigens, and only after the appearance of mature virus. The successful application of the use of two immune sera, each labeled with a different fluorescent dye for the simultaneous visualization of two antigens within a cell, is reported. Using this technique, the sites of LS and NP antigen synthesis, were easily differentiated. The intimate mixing of the two antigens at a later stage appears to coincide with the fragmentation of the inclusion body and the first detectable increase in cell-associated virus. The evidence obtained strongly suggests that the typical inclusion body observed in vaccinia-infected cells is composed mainly of the NP antigen.