In earlier papers hitherto published on this serial study, the author and the co-workers have reported that the Japanese B encephalitis virus can easily multiply in Ehrlich ascites tumor cells propagating in mice. Furthermore, they reported that the infection of these tumor cells with the virus could be established in a moment, and once the first step of the infection was accomplished, the cell infected virus instantly enters such a state, that it can not easily be neutralized by the hyperimmune serum of enough neutralizing potency against the virus, although the virus existing in a free state can readily be neutralized by the same serum. Such an infected virus can persist in a latent state for considerably a long period of time in the tumor cells that are existing in mice which contain the neutralizing antibody of significant potency.
In the present study the author performed experiments in order to investigate whether the infection of cells with the virus can also be established in a case as such, that the cells had previously been in contact with the neutralizing antibody for a certain period of time, and afterwards encounters with the virus that comes into this environment.
Experimentations are as follows; i.e. 1.0ml. of undiluted hyperimmune rabbit serum against Japanese B encephalitis virus and 1.0ml. of packed mass of washed Ehrlich ascites tumor cells of mouse were mixed and incubated at 37°C for 30min. After the incubation, to each of these mixtures was added each of decimal dilutions of Japanese B encephalitis virus, from 10^-1 to 10^-7 in amount of 1.0ml. respectively. The mixture consisting of the antiserum, the cells and the virus, was again incubated at 37°C for 30min. After that, the mixture was centrifuged. The neutralization of the virus existing in free state in the mixture of the respective virus dilution was testified by the direct intracerebral inoculation of the supernatant of each of them to mice. The cell adsorbed virus in each virus dilution was put to the demonstration test by the indirect in vitro cell virus infection method, described as follows. The cells in the sediment were washed three times and were inoculated each to three normal mice in certain amount intraperitoneally. Four to 7 days thereafter, the tumor fluid of the mice of each group was drawn, pooled and centrifuged respectively. The supernatant was put to the demonstration test of the virus with the direct method of mouse intracerebral inoculation. When the result of this was positive; i.e. if the virus was demonstrated in the ascitic fluid, the cells that had been intraperitoneally inoculated 4-7 days ago, were designated to have been infected with the virus, although the trials of demonstration of the free virus in the same respective mixtures had resulted in negative.
The results are as follows; i.e., the cell adsorbed virus could be recovered by the indirect method from the mixture consisting of the cells, the virus and the immune serum of rather surplus potency, while the free virus could never be recovered from it by the direct method. In a word, it was demonstrated that the Japanese B encephalilis virus could be adsorbed to the Ehrlich ascites tumor cells that had been suspended in immune rabbit serum or immune mouse ascitic fluid of sufficient potency against the virus and had been in contact with the antibody, and could establish the first step of the virus infection of the cells in this environment.
There remains much to be made clear as to the mechanism of this phenomenon, but standing upon the results obtained so far, it may be possible to conclude, that the first step of virus infection of cell can be established in the existence and under the action of the neutralizing antibody against the virus, so far as at least the Japanese B encephalitis virus and the Ehrlich mouse ascites tumor cells are concerned.