1. The production of the neutralizing antibody as well as the antihemagglutinin were investigated with the course of the intracutaneous inoculation of vaccinia virus in rabbits, comparing with the histomorphological changes in the inoculated loci with the consideration of their primary and secondary infections.
2. The neutralizing antibody reaction is more sensitive than the anti-hemagglutination reaction both in the primary and the secondary infected rabbit, although the course of the changes are nearly equal as a whole with each other.
3. The first appearance of the neutralizing antibody and the antihemagglutination reaction was noticed since the sixth day of the primary inoculation, slightly later than the plasma cell reaction in the infected skin (4th day). On the 8th day, there appears a hemorrhagic reaction in type of the Arthus-phenomenon in the inoculated skin, in accordance with which the titer of both antibodies fall transitorily, followed by the abrupt rise there after.
4. The secondary inoculation was given to those rabbits, by which the local changes in type of the Arthus phenomenon (so-called rapid reaction in Piquet's concept) appeared promptly, accompanied with the fall of the titers of antibodies. However, in this case the recovery of the antibodies was reached since the third day. Moreover, on the 7th day the plasma cell reaction appears in the infected locus with the additional formation of the antibodies in that rabbits.
5. From those transitory fall of the antibodies (neutralizing and anti-hemagglutination antibodies) accorded with the appearance of the reaction in type of the Arthus phenomenon and from the simultaneous disappearance of the viremia, we can assume an antigen-antibody reaction as a basis of those reactions in the inoculated rabbits.

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Concerning the conditions for the hemagglutination reaction of sheep red cell by Col-SK virus, various experiments were carried out. The method of hemagglutination test was as that used for Japanese encephalitis. As antigen mouse brains infected with Col-SK virus by intracerebral inoculation were made into 10% emulsion with Ringer's saline and centrifuged at 3000-5000r.p.m. for 20 minutes. For blood cell, 0.25% suspension of sheep red cell was used. Qualitative differences between the samples of erythrocyte of individual sheep were great so that about a half of the samples failed to agglutinate. The antigen loses its avidity for hemagglutination after heating at 37°C for 3 hours, or at 56°C for 30 minutes though its infectivity to mouse remained. The avidity for hemagglutination and infectivity do not go in pararell. In general experiments Ringer's saline was used for diluent. In hemagglutination reaction monovalent cation plays an important role. When erythrocyte suspension (NaCl as diluent) is added to each serially diluted antigen (NaCl as diluent both for antigen and serial dilution), agglutination titer is rather low, whereas KCl and LiCl isotonic solution are used in place of NaCl, the highest titer obtainable is observed as the result. Even a very little addition of KCl, LiCl or CsCl in dose of 1/1000-1/100M show high agglutination titer. NH₄Cl, CaCl₂ and MgCl₂ failed to demonstrate hemagglutination reaction. When NaF is added to Ringer's saline in dose of 1/100M no disturbing effect is observed. Eighty seven % of agglutinin was adsorbed at 4°C 120 minutes. A certain part of agglutinin thus adsorbed was dissociated at 37°C.

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Hemagglutination inhibition test by Col-SK virus for the sera of the healthy and the poliomyelitis patients collected from various parts of Japan was carried out. In human serum there exist, as a rule, hemagglutinin for sheep red cell, which is eliminated by the addition of 10 times amount of 10% red cell suspension for 15 minutes at room temperature. In the sera of the healthy population inhibitory substance against hemagglutination of sheep red cell by Col-SK virus was not recognized.
Hemagglutination reaction by Col-SK was not inhibibited by the immune sera of each type of poliomyelitis (Brunhilde, Lansing and Leon) and also of Japanese encephalitis and some other diseases. In animal experiment the inhibition antibody was confirmed with the SK virus infection.
Of 324 serum samples of the healthy Japanese collected from various parts of Japan, 24 samples (74%) were positive. Speaking from the geographical viewpoint, they are mainly from Kumamoto and Hachijoisland. Of the sera from the Japanese mainland and Hokkaido very rare found positive. As for the age distribution of the positive cases, under 2 year old group shows higher positive rate than that of over 2 year old group. Samples of 167 polio cases and 45 cases suspected of non-paralytic polio were all negative. It is presumed, that no cases among the polio and the suspected polio patients in Japan could be ascribed to Col-SK origin.
Neutralization test with 75 samples of the healthy Japanese in various parts of Japan shows that only 2 samples from Kumamoto are positive.
From the above mentioned results this may be concluded that Japan has already been disseminated by Col-SK virus and also that the grade of its dissemination might increase higher towards the southern part of the country such as Kumamoto and Hachijo island.

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The contents of glutamic acid in the infected brain of mice with Japanese B encephalitis virus were compared with that of the healthy mice.
1) The contents of glutamic acid in the infected brain of mice were 215mg. % and showed the increase of 45 per cent compared with the healthy one.
2) The gradual increase of glutamic acid in the infected brain of mice was obserbed as the course of the virus infection proceeded.

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“IZUMI Fever” is one of the important exanthematic diseases in this country, which is confined almost to children and may occur either in a sporadic or in an endemic form. It is characterized by a fever of a saddleback type whose onset portends an appearance of a transient fine rash resembling that of scarlatina and followed by some intestinal disorders.
Many authors have endeavoured to isolate the virus and to establish their causative nature, but almost all of them failed in volunteer experiments. In this respect we succeeded to produce a typical infection in an 8-year-old female by an oral administration of the isolated virus. After 4 days incubation period, saddleback type fever, bilateral exanthemata of arms, angina and urobilinogen in urine were recognized and the study is now in progress to isolate the same virus from feces and blood of this child again.
The virus was isolated from a sporadic case, by intracerbral inoculation of the Berkefeld-N filtrate of feces to young mice. Subsequently the virus was transferred from brain to brain and now fully adapted to mice. These mice usually died until 5 days after inoculation and LD₅₀ for mice weighing 9g. in average is 10^-8.5. By intracerebral inoculation to mice the virus distributes in brain, liver, spleen, lung and kidney and intranasal and intraperitoneal administration also cause death of mice. They are stored in 50% glycerol for 4 month without loss of activity.
Histopathological changes in mice was limited at the beginning of isolation of the virus rather to liver, spleen and kidney but later chiefly focused in brain, causing encephalitis and leptomeningitis. Egg culture and purification of virus were performed, but not yet succeeded.
Complement fixation test was also undertaken. The antigen was prepared by Casal's method from the brain containing this virus and ASK₁ (isolated by Kitaoka and others). Immunization of guinea pigs with one of both strains proved the elevation of C. F. antibody against both antigens. Therefore a cross immunity was proved in C. F. T. between these viruses, but not in neutralization test. Neutralization antibody against this virus was proved in the serum of patient from whom the virus was isolated 3, 4 and 6 month after onset, while no antibody was proved in these sera against ASK₁. The sera of the experimentally infected patient mentioned above, 2 and 4 month after onset also possessed the antibody.
Some disscussions were made about the tropism of this virus.

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Evaluation experiment of the Glutamyl cholin and V. B₁ treatment was carried out using cynomolgus monkeys (body weight ca. 1.5kg). The viruses of poliomyelitis Lansing and Brunhilde strains were intracerebrally inoculated. When the infection was clinically confirmed by the onset of fever or paralysis, the treatment was immediately started. GLC 1cc 3mg mixed with V. B₁ 1cc 5mg was given daily by intrathecal injection.
The experiment for the Lansing strain challenged group was not successful. Five monkeys inoculated with the virus in dilution of 10^-2 and 10^-3 died and only one monkey survived. All three monkeys for the control died of the dsease with no exception.
Brunhilde strain was also challenged in the same way. The groups inoculated with the virus in dilution of 10^-2 and 10^-3 received the GLC and V. B₁ treatment according to the above mentioned method. Three monkeys out of 9 (4monkeys are of 10^-2 dilution group and 5 of 10^-3 group) survived after the treatment.
Though it should be carefully refrained to conclude that the lower mortality rate in the treated group is statistically significant, close observation about the duration. and the clinical course of the disease implies a favorable prognosis of the disease, supporting the effectiveness of the treatment. When the harmonic means of the days elapsed from the onset of the clinical symptoms to the death of the animal were compared between the control and the treatment groups a noticeable prolongation of the days are recognized among the latter group. Also studying the fever curve and the course of the disease some monkeys of which the symptoms were all serious with the critical fall of body temperature below 35°C were found recovering (though paralysis remained) in the course of the treatment which lasted more than one month. So far in our experiment, monkeys intracerebrally challenged with poliomyelitis viruses and had the body temperature fallen below 35°C died unexceptionally when without GLC and V. B₁ treatment.
All the observations from the various angles indicate that the treatment may be recommended as effective when it is adequately started from the early stage of the disease.

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I have already made experiments on the interference phenomena of Japanese B. Encephalitis virus on the growth of Influenza virus in the developing chick embryo. In my present experiment on the interference phenomena of Japanese B. Encephalitis virus and the Newcastle disease virus in the developing chick embryo, I have found that as was with the Influenza virus, the pre-inoculation of the Japanese B. Encephalitis virus 72 hours beforehand also suppressed the propagation of the Newcastle disease virus. Various other factors affecting the interference phenomena of the two viruses have also been discussed. The growth of the Japanese B. Encephalitis virus in the developing chick embryo may be detected by using the Newcastle disease virus as an indicator.

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I have observed that the Japanese B. Encephalitis virus interferes with the growth of the Mumps virus in the developing chick embryo. Checking the various factors affecting this phenomena of interference have led me to the belief that the growth of the Mumps virus is interfered by the Japanese B. Encephalitis virus in the similar manner as in the case of Influenza virus or the Newcastle disease virus.
The results of the three series of experiments have led me to the conclusion that the viruses of Influenza, Newcastle disease and Mumps are all interfered by the Japanese B. Encephalitis virus in essentially the same way. A proposition have been made by previous workers of placing these three viruses in the same group on the ground that they all share the ability to agglutinate the red blood corpuscles and also from other similarities. I also would like to advocate the grouping of the three from the standpoint of their similarity in regard to the phenomena of interference.

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Strains used were Enders strain and freshly isolated Fujimura strain which was carried through 16 serial transfers in the amniotic sac.
The amniotic fluid harvested 5 days after inoculation of 7-day-old embryonated eggs through amniotic route was purifid by procedures given in the table. All manipulations were carried out in the cold.
The final yield of haemagglutinative factor was 50% of the starting material and the protein test by sulfosalicyl acid was negative.
Agreeing with the first report of the electron microscopy of mumps virus by M. L. Weil, D. Beard, D. G. Sharp and J. W. Beard, most of mumps virus particles appeared poor in electron scattering power resulting in pictures of very low contrast (Fig. 1), and therefore, Cr shadowing by the method of Williams and Wyckoff were. used for the most part. Micrographs were done with the JEM electron microscope.
As can be seen in the table, the crude viral suspension was purified partially on purpose of yielding every particulates present in the infected amniotic fluid, because we want to attempt to get a clue to the developmental stages of the mumps virus, which may be seen on electron microscope examination of partially purified specimens, other than the attempt of discernment of morphological differences between samples derived from infected materials at different stages of infection.
The micrographs under these conditions revealed three different natures of particle kind.
a) LARGE PARTICLES OF IRREGULAR SHAPE:
They appeared exceedingly variable in size ranging from 120 to 260mμ, but they were equally flattened and roughened, some had a pitted area showing doughnut-like structure. These characteristic features of the particles agree with those of mumps virus tudied by Weil et al.
Fig. 4 shows large aggregates of the particles which possibly due to the use of saline only as a medium for redispersion.
b) MEDIAN SIZE PARTICLES:
They appeared circular and considerably homogeneous in size ranging from 60 to 75mμ (Fig. 8). At the present time nothing can be said about the nature of these particles.
c) SMALL PARTICLES:
The micrographs shown in figures 2 to 6 reveal small spherical particles uniform in size and shape (approximately 20mμ in diameta) in profusion. Some of them, maybe not all of them, may represent the “normal compont” particles in the amniotic fluid, which nobody revealed electron microscopically so far. Especially note the closely packed aggregates of small particles. (Fig. 7)
d) In Fig. 11. extremely long continuous structures originated from mumps virus particles are noticeable. The lengths and widths vary widely. The electron micrographic characters of the structures look to reveal quite similar to those of nucleic acid reported by J. F. Scott. (Biochimica et Biophysica acta, 2, 1-6, 1940) It is considered likely that they represent a lateral and longitudinal aggregation of nucleic acid released from the unstable mumps virus particles.

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