The intratypic serodifferentiation test of types 1 and 2 polioviruses was investigated after Wecker's method. Variables in the test were examined extensively with type 1 polioviruses; these included animals to be immunized, immunization schedules, time of bleeding, temperatures of incubation for the test and criterion of the evaluation of the results obtained. It was proved that the modified Wecker test was applicable to the differentiation of Sabin vaccine-related types 1 and 2 poliovirus strains from wild poliovirus isolated in Japan before the mass vaccination with Sabin vaccine in 1961. It was confirmed that the vaccine-related strains showed antigenic drift from the original strains during human passages. Several problems concerning the serological differentiaton of polioviruses are presented and discussed.
The complement-fixing tumor (T) antigen induced by simian virus 40 (SV40) has been prepared from SV40-infected cell cultures, from infected cell cultures treated at the time of infection with 1-beta-d-arabinofuranosylcytosine (ara-C), and from SV40-transformed cells. Upon partial purification, the T antigen exhibited the following properties: it was tightly adsorbed by calcium phosphate gel, it was precipitated by acetic acid at pH 5 or by ammonium sulfate at about 20 to 32% saturation, and it had a molecular weight greater than 250,000, as estimated by Sephadex G-200 gel chromatography. In contrast, deoxycytidylate (dCMP) deaminase, thymidylate (dTMP) kinase, and thymidine (dT) kinase were less strongly bound to calcium phosphate and were not precipitated at pH 5; these enzymes also had much lower molecular weights than the T antigen, as did dihydrofolic (FH(2)) reductase. Furthermore, higher ammonium sulfate concentrations were required to precipitate dCMP deaminase, dTMP kinase, and FH(2) reductase activities than to precipitate the T antigen. Another difference was that the T antigen was not stabilized, but dCMP deaminase, dTMP kinase, and dT kinase, were stabilized, respectively, by dCTP, dTMP, and dT or dTTP. Deoxyribonucleic acid (DNA) polymerase activity resembled the T antigen in adsorption to calcium phosphate, in precipitation by ammonium sulfate or at pH 5, and in the rate of inactivation when incubated at 38 C. However, the polymerase activity could be partly separated from the T antigen by Sephadex G-200 gel chromatography. The cell fraction containing partially purified T antigen also contained a soluble complement-fixing antigen (presumably a subunit of the viral capsid) which reacted with hyperimmune monkey sera. The latter antigen was present in very low titers or absent from cell extracts prepared from SV40-infected monkey kidney cell cultures which had been treated with ara-C at the time of infection, or from SV40-transformed mouse kidney (mKS) or hamster tumor (H-50) cells. The T antigen, however, was present in usual amounts in SV40-transformed cells or ara-C treated, infected cells.
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The synthesis of papovavirus SV40 tumor (T) and virus (V) antigens in infected green monkey kidney cell cultures incubated at different temperatures was studied. At 37°C or higher (up to 43°), both T and V antigens were formed reaching peak titers in 2 to 4 days. However, at 23°C, there was hardly any development of V antigen, while the T antigen reached the same peak titer obtained in cultures incubated at 37°. The 23° cultures with peak amounts of T antigen appeared normal, but raising their temperature to 37° was followed by a rapid hundred-fold increase in V antigen and the appearance of cytopathic changes. These findings indicate that the synthesis of tumor antigen is less temperature dependent than is the synthesis of virus antigen. The thermal separation of the synthesis of tumor and virus antigen confirms the finding obtained previously with DNA inhibitors—that is, formation of tumor antigen is not dependent upon the synthesis of virus.
The effect of DNA antagonists and various antibiotics on steps in the synthesis of SV40 virus in green monkey kidney cells was investigated. Both the early forming tumor (T) antigen, as well as the later synthesized virus (V) antigen, were synthesized in the presence of fluorouracil and iododeoxyuridine. Cytosine arabinoside (and fluorodeoxyuridine in starved cells) prevented synthesis of V antigen but not T antigen. The synthesis of T antigen therefore does not require synthesis of virus DNA. Virus particles formed only in the presence of the iododeoxyuridine and they were non-infectious. Actinomycin D inhibited synthesis of both tumor and virus antigens, suggesting that the synthesis of these antigens involves DNA-dependent RNA. Puromycin allowed synthesis of the T antigen which remained localized at the nucleolar membrane. This finding with puromycin suggests that the T antigen is a protein of low molecular weight. Virus antigen forming in the presence of mitomycin C, p-fluorophenylalanine, iododeoxyuridine, or fluorouracil was distributed atypically. These inhibitors caused the V antigen to be diffusely spread throughout the nucleus, or to be concentrated at the nuclear membrane.
Cells infected with the papovavirus SV40 not only synthesize viral antigen but also synthesize the specific nonviral antigen found in SV40-induced tumors. In the presence of the DNA antagonist cytosine arabinoside, infected cells fail to make viral antigen but still synthesize the tumor antigen. Iododeoxyuridine does not inhibit the synthesis either of tumor or of virus antigen but does prevent the development of infectious virus.
Cell cultures derived from various types of human cancers were examined for the presence of tumor specific antigens by both immunofluorescence and complement fixation methods, in which the serum of the patient was tested against cells grown from his cancer. The methods, previously used successfully to detect a new intranuclear antigen induced by the papovavirus SV40 in transformed human and hamster cells, and in hamster tumors, failed to reveal the presence of immunologically reactive components.
