In making virus inoculation experiments, it is essential to keep the susceptibility of the animals in a constant level.
There are many factors which determine the susceptibility ; especially in case when the intraperitoneal inoculation of Jacan ese B encephalitis virus is made, postnatal date and body weight of the mice used are important in determining the susceptibility.
The present author examined these factors along with the influences of room tem-perature. It is also assumed important in elucidating the cause of changes of susceptibility to confirm whether the difference in body weight has influence on the rise and fall of the virus population after intraperitoneal inoculation. The results were as follows:
1) The postnatal date of mice has the close connection with their body weight; the 3-week-old one weighed 8 g, the 4-week-old 10 g, the 5-week-old 15 g on the average.
2) Susceptibility of mice to the virus intraperitoneally inoculated decreases as their body weight increases, but there is no definite interrelation between fatality rate and a challenge dose of the virus, in the case of mice over 15g.
In the mice of body weight from 8 to 10 g, susceptibility is considerably high and it draws a parallel line between the onset and the virus dose inoculated.
Therefore, it is preferable to use the mice of 8 to 10 g for the intraperitoneal chal-lenge of the virus.
3) Susceptibility to the intracerebral and intraperitoneal inoculation of Japanese Bencephalitis virus of the mice which were raised in the high room temperature for a long time is higher than that of those bred in the low room temperature.
4) It is after 48 hours that the virus is recovered from the brain of mice weighing 8 g which was intraperitoneally inoculated with the 10-1 dilution of the infected brain. And 98 hours after the inoculation, recovery of the virus attains the uppermost amount, then the virus nearly corresponds in potency to that from intracerebral inoculation.
In the brain of the 20 g mice, even if the attack is not manifest, no small amount of the virus is demonstrated though less in comparison with that of 8 g mice.
The virus is detected in the blood of 8 and 20 g mice in high concentration up to 12 hours after the challenge, null 48 hours later, and 72 hours later again demonstrated. Thereafter no virus is detected.

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In order to make clear the quantitative relationship between virus and neutralizing antibody the distibuton of neutralizing antibodies in brain and serum after intraperitoneal inoculation of the immune sera into mice, was examined by protection test and neutralization tests of serum and brain emulsion.
The results were as follows:
1) Inhibiting effect upon Japanese B encephalitis virus of goat's immune sera intraperitoneally inoculated to the mice was investigated at 4 times of 3, 6, 10 and 14 days after the inoculation.
a) Protection against the intracerebral challenge reaches the height 6 days after the inoculation, and then declines sharply.
b) Neutralizing antibodies in the mouse blood reach their maximum amount 3 days, after the inoculation, there after turn to the rapid decline.
c) Neutralizing antibodies in brain reach the maximum 6 days after the inoculationand then promptly diminish to reach the minimum.
In the brain of normal mouse of 10 g, not the least antibodies are demonstrated.
2) It appears that protection against the intracerebral challenge runs parallel with the amount of intracerebrel neutralizing antibodies.
3) Transmission to the brain of the neutralizing antibodies contained in immune sera intraperitoneally inoculated is much restricted in the quantity and time, and the disappeararce of antibodies is facile as in the case from the blood. The amount of the intra-cerebral antibodies is either equal to or less than that in the blood.
This fact was discussed on the basis of “Bloop-Brain Barrier”.
4) Validity of passive immunization is maintained 2 weeks long or so when 0.5 cc of goat's immune sera with neutralization index of 13, 000 are administered intraperitoally to the mice.

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Bovine strain rinderpest virus was adapted on embryonating eggs by the inoculation repeated first on the chorio-allantoic membrane and then into the yolk-sac (avianized rinderpest virus-YS strain). This YS strain was further adapted by the inoculation into the vein of chorioallantoic membrane (avianized rinderpest virus-IV strain). Biological nature and the mode of infection on the embryonating eggs and cattle were compared between the two variants YS strain and IV strain. Various tests were made, further, on the preservability of freeze-dried IV strain as well as on its immunogenici1y as vaccine.
1. When inoculated into the vein of chorio-allantoic membrane, IV strain showed stronger intensity of infection and lesions than that of YS strain . When the inoculation was made into the yolk-sac, however, there was no difference between the two.
2. In either case of the inoculation made with IV strain intravenously or into the -yolk-sac, no difference of mortality was found in chick embryos.
3. Pathogenicity of IV strain on Japanese black cattle was much weaker than that of the original YS strain and the inoculation reactions were negligible.
4. As the minimum infective dose (MID) of IV strain on cattle parallels that of chick embryos, the amount of virus vaccinated can be determined by the determination of its chick embryo MID.
5. Appearance of viremia is inconsistent in the cattle infected with IV strain and even when it develops, it disappeares within a short period of time. On the contrary, when infected with YS strain, viremia appears more frequently with longer duration. When infected with bovine strain or rapinized strain, moreover, viremia appears in 100% with much longer duration.
6. Virus titer becomes highest when IV strain is inoculated intravenously on the embryonating eggs of 11-12 days and incubated for 5 days and also when the same strain is inoculated into the yolk-sac of the embryonating eggs of 5 days and incubated for 7-9 days. The former, however, gives a titer slightly higher than the latter.
7. Virus content of the infected egg reduces rapidly following the death of embryo.
8. Cattle inoculated with IV strain are completely immune against the challenge with highly virulent bovine strain and this immunity lasts at least for 1 year.
9. Cattle inoculated with IV strain inhibit the challenge of bovine virus in such an early stage of 4-5 days after inoculation.
10. Complement fixation antibody of the cattle infected with IV strain is produced quicker in proportion to the amount of virus inoculated, but the highest antibody titer is little influenced by the amount of virus inoculated.
11. Investigations were made on the preservability of freeze-dried IV virus in order to use it as vaccine. It maintains the chick embryo virus titer of 10-3 for 1 year and. over when preserved at -40°C, 9-12 months at 0-5°C, 1-3 months at 22°C and 1-2 weeks at 37°C. When once dissolved, it maintains the same titer for 6-9 hours at 22°C and only for 3 hours at 37°C.

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Neutralization, complement fixation (CF), and hemagglutination inhibition (HI) testsare commonly applied as seroreaction of Japanese encephalitis (JE) with human serum. With domestic animals, however, the neutralization test mainly has been performed until this time. The present study was carried out to apply the CF and HI tests for serum of demestic animals, such as horse, bovine, swine, gout, fowe, and pigeon.
In the mouse brain infected with virus, the proliferation of viruses, the appearance of CF antigens and HI antigens run parallel each other, and there are the closed relation between the titer of CF test and that of HI test. However, CF antigen could be differentiated from HI antigen by absorption with red blood cells or by heating treatment. From this point of view, the antigens and antibodies concerned with these seroreactions were investigated in this study. The comparative study of antigenicity of original viruses with those of chicken embryo culture and hamster tissue culture, also were carried out.
1. The HI test for serum of domestic animals could be successfully carried out by treating serum with acetone and by absorbing with red cells, The CF test by common inactivation for serum of horse, goat or pigeon, but for that of swine by at 60°C for 30 minutes and for that of fowl by treating with aceton in low temperature.
2. The HI antibody was remarkably decreased by absorption with pigeon red cells coated with a large amount of the HAin, but the CF antibody hardly decreased. On the contrary, by absorption with material containing a large amont of the CF antigen, the CF antibody diminished markedly, while the HI antibody, hardly.
3. In pigeons and mice inoculated with the pigeon red cells coated with the HAin, the HI antibody was easily produced, but the CF antibody hardly.
4. No differences of antigenicity for CF and HI reactions were recognized between original viruses and those of 70 generations passage in one day developed chick embryo and of 20 generations passage in hamster kidney tissue culture.

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