In the previous paper we reported the isolation of SG strain of Izumi fever virus which was isolated from feces of a sporadic case with successful humann experiments by the feeding. The present paper deals with the isolation of a similar virus from a patient in an epidemic outbreak, in which about 200 children suffered in total. In April, last year (1953), an outbreak of typical Izumi fever occurred at Mizonobe Village, Yamagata prefecture. At the time, the isolation of virus was performed with the same procedure described in the previous paper and a similar strain of virus (MYC) was obtained from one patient. The virus was also adapted to mice brain by intracerebral transfers, and as the result all of the findings, such as signs of disease in mice, histopathological changes and so on, was quite similar to those observed at the first isolation of the SG virus. At the beginning of isolation, the histopathological changes were almost limited to the liver, lung, spleen and kidney of mice but after the adaptation by intracerebral route, the changes became conspicuous in the brain causing an encephalitis. Further the serological examination indicated the cross reaction between both viruses.
In addition, the supplementary work of the first report was described concerning about the reisolation of virus from volunteer patient. Though the reisolation was unsuccessful, several incidences at the primary isolation of SG strain was again experienced at the reisolation. That is, the histopathological examination of the sacrificed or dead mice of the 2 or 3 transfers at the beginning, revealed the characteristic visceral lesions of Izumi fever. From the facts mentioned above in combination with the results of volunteer experiment and of neutralization and complement fixation tests, it may be concluded, that the SG strain will be probably an etiological agent of Izumi fever.

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In the brain tissues of the guinea-pigs, immunized against the Japanese B encephalitis with the intracerebral inoculation of the active or inactivated virus, the intracerebral concentration of the neutralizing antibody can be observed in loco. Namely, certain period of time after the intracerebral or subcutaneous antigen inoculation, the brains of all the animals were perfused with saline and the neutralizing antibody contained in the perfused brain tissues, excluding the participation of the serum antibody of the circulating blood in the brain, was titrated, and it was ascertained that the perfused brain tissues of the intracerebrally immunized animals contained the antibody in higher titer than those of the subcutaneously immunized ones, even when the serum antibody is present in nearly the equivalent titer in the circulating blood of the animals in both the intracerebral and subcutaneous immunization groups. In regard to this fact, the following two phenomena may be able to be considered as the substantial cause of it, namely, in the first place, the local intracerebral antibody production in situ, and secondly, the permeation into and the adherence to the brain tissue of the serum antibody of the circulating blood. In the present study it was elucidated that the titer of the antibody in the brain tissue rises significantly either later than or at least concomitantly with and not at all earlier than that in the serum of the circulating blood. It was also made clear, by the antibody titration on the perfused brain tissues, that after the repeated intracerebral immunization the antibody in the brain tissue rises and falls utterly in accompaniment with that in the circulating blood, and also that the former is present as long as the latter is present and disappears if the latter disappears. Standing upon the results described above, it might be possible to consider that the substantial causes of the intracerebral antibody concentration is principally the permeation and adherence of the serum antibody to the brain tissue, owing to the rise of permeability of the blood-brain-barrier caused by the brain injury such as the intracerebral antigen inoculation.

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From the quantitative analysis on the clonal distribution of plaque type mutants of T 2 L phage, Luria has led an interesting hypothesis on the mode of replication of virus genes. That is, the replication rate of the genetic determinants will be exponential if each replica acts as a source of new replicas. Assuming the constant probability of mutation per individual genetic markers, therefore, mutant phage will appear clonally in the host cells with the frequency which is inversely proportional to the number of mutants.
The present paper represents some experiments designed for testing the generalization of Luria's theory using another bacteriophage system with respect to the another type of mutation, i.e., host range mutation. The bacteriophage system used here, P6 phage and its host Escherichia coli, E6 strain, is suitable for analysis on the host range mutation because of its high mutation rate (10^-6).
The experimental results described in this paper showed clearly that Luria's theory holds for our bacteriophage system as to host range mutation indicating that the multiplication of vegetative phage particles of P6 is likewise exponential.

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1. An outbreak of epidemic myalgia prevailed among the employees in a company in Kawasaki in the summer of 1952. Many of the patients developed the clinical symptoms typical for epidemic myalgia but the remainders showed only fever and other uncharacterized symptoms.
2. Precise clinical descriptions were made of some of the cases. They were almost typical for the usual epidemic myalgia.
3. A strain of B group Coxsackie virus was isolated from a patient among them. This strain was serologically proved to belong to Bl of the classification by Dalldorf.
4. It is very probable that this outbreak was caused by this type of Coxsackie virus.

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Inoculation of trachoma material into the testicle of guinea pig gave, in serial passage, a positive result in 9 strains out of 31 ones. That is, an orchitis occured respectively. In the majority of cases the inflammation of testicle was not so serious: the tunica vaginalis and the parenchyma were slightly hyperemic and edematous.
The inflammatory testicle is excised, ground down sufficiently and suspended in saline solution. The suspension is frozen and melted in freezing mixture several times, and after it is centrifuged at 4, 000 rpm for 30 minutes, the supernatant fluid is employed as antigen.
The agglutination tests were performed in 37 cases of trachoma and 20 cases of non-trachoma. The tests proved positive in 20 cases (54%) of trachoma and 1 case (5%) of non-trachoma. If explained in detail, in 31 cases of stadium I and II of trachoma 26 cases (64.5%) were positive, in 29 cases of stadium I 19 cases (65.5%) positive, in 20 cases of granular tarchoma 16 cases (80%) positive.
Therefore, it is the author's judgement that the agglutination is of specific reaction for the serum of trachoma patient.

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The susceptibility to virus should be reconsidered by the specificity of virus different from other bacteria. By conducting a study on the effect of Cortisone on the susceptibility of mice to virus, I have confirmed that the administration of Cortisone increases the susceptibility of mice to the mouse encephalomyelitis virus (GDV11 strain). For this purpose it is necessary to administer comparative large doses of Cortisone before inoculation of virus. On that occasion the effect of Cortisone in the groups of intraperitoneal or subcutaneous inoculation of virus is more significant than that in the groups of intracerebral or intranasal inoculation.
This experiment is thought to be suitable for analytic study on the problems of susceptibility to virus. The essential of this effect will be reported on later date.

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Employing deembryonated eggs, H. Bernkoph (1949-1950) has succeeded in cultivating influenza virus in a similar manner to the tissue culture. The authors, attempted to cultivate Japanese B encephalitis virus (Nakayama strain) by the modifications of the above method.
All of the contents, except the chorioallantoic membrane, of the fertilized eggs incubated for 12 to 15 days were removed from the egg shell, and the eggs were refilled with the medium mainly composed of 2% yolk saline solution. In addition, Japanese B encephalitis virus was inoculated into the eggs in an amount to hold the virulence at 10^-3, and the eggs were incubated at 37°C. The following is the results thus obtained.
Better results in the multiplication of the virus were obtained with the developing eggs incubated for 13 or 14 days when compared with those obtained with the eggs incubated for 12 days or for 15 days. The LD₅₀ determined in the former cases were 10^-6.0 and 10^-5.8 respectively, while in the latter, they were 10^-5.5 and 10^-4.6 respectively.
As to the incubation period of the culture, the multiplication of the virus was found more favorable when incubated for 24 hours or 48 hours than 72 hours, showing the LD₅₀ values at 10^-6.0 and 10^-5.5 respectively and 10_-4.5 for the last case.
When 100-1, 000units/cc of the medium of penicillin was added, an increase in the LD₅₀ value was recorded showing the highest value in the LD₅₀ at 10^-7.0.
Further, the highest LD₅₀ value attained by the addition of 10% extract of the developing chick embryo (7-11 days old) at the rate of 1% of the medium was 10^-7.8, and the average was 10^-7.2.
All of the above experiments were conducted with the eggs not infected by Japanese B encephalitis virus. With the eggs infected by Japanese B encephalitis virus, almost identical results, though slightly lower in the LD₅₀ values, were obtained.

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1) Most of bacteriophage P7 particles liberated from host bacterium (Sh. dysenteriae type II) by massive lysis, combine with bacterial debris and are inactivated. This inactivation proceeds gradually during preservation at low temperature.
2) This attachment is reversible and combined phage dissociates by heating, but in order to acquire maximum dissociation, it is necessary to dilute the phage-stock before heating. Some examples of the relationship between time and temperature to acquire maximum dissociation are as follows: 2 minutes above 50°C, 20 minutes in 43°C and 2 hours in 37°C.
3) Being unable to use as material of experiment the phage-stock containing phage that is reversibly inactivated, we removed the debris from lysate by centrifugation at 10, 000 R. P. M. for 20 minutes after heating 10 minutes at 63°C.
4) We discussed the above phenomenon comparing with other phage-systems of stable titer, and inferred that the attachment of phage to the bacterial debris might probably be caused by phage-receptor, left on the bacterial debris.
5) Pleomorphism of plaque size is related to this reversible inactivation of the phage.

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Bacteriophage P7 combines reversibly with the debris of bacteria, lysed by itself. Though this combined phage is dissociated by heating or by diluting with distilled water, 0.1 Mol NaCl, or 0.01 Mol MgSO₄, it is not dissociated by diluting with 0.01 Mot MgCl₂ nor 0.01 Mol CaCl₂. Moreover, heating leads to dissociation in case of 0.01 Mol MgCl₂ as in broth, but in case of 0.01 Mol CaCl₂, heating is of no effect.
These characters of dissociation are quite similar to the characters of dissociation in the attachment between heat-killed bacteria and the bacteriophage P7, therefore it seems to be a specific attachment between the phage and phage-receptor which is left on the bacterial debris.

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Japanese encephalitis is confirmed to be distributed not only in Japan, but also in more wider area, which extend from South Manchuria and Maritime Province to Burma and Java. The northern limit of the distribution of J. E. in Japan was suspected to exist in Hokkaido island. So we tried to make clear the distribution of J. E. in Hokkaido.
Neutralization tests against J. E. virus were made with sera of 642 persons and 587 horses collected in Hokkido island. These persons and horses were selected among those, who were born there, had never made a trip to other district, and had never received J. E. vaccine injection.
By the results of these neutralization tests, northern and most eastern part of Hokkaido were proved no sera positive (Fig. 1). This area is identified as non-epidemic area (area D), where found no evidence which proves virus existence.
Case reports of J. E. and Japanese equine encephalitis (J. E. E., which is proved to be produced by the same virus of J. E.) in Hokkaido were surveyed. Especially, the epizootic in 1948 was the far greatest. Even by this epizootic, no cases were reported in eastern part of this island, added to the non-epidemic area (Fig. 2). So this area, where neutralizing antibody to be proved positive and no cases ever reported is identified as sub-epidemic area (area C). About 8% of human sera (20/246) and horse sera (21/251) collected in the C area were proved neutralization antibody positive (Tab. 1).
Morbidity rate of J. E. E. in each age group of horses is statistically studied. In area B, no difference was noticed between the morbidity rates of young and aged groups. (Tab. 3). But in area A, much difference was noticed. In aged group in area A, the morbidity rate is clearly diminished. It may show the development of protective immunity in aged group. Area B, where no protective immunity is noticed, is identified as temporary-epidemic area. Area A, where the existence of protective immunity among aged horses is proved, identified as epidemic area.
These gradual progression-development of neutralization antibody among horse and human being → outbreak of epizootic → development of protective immunity in aged horses - must be resulted by the gradual increasement of viral invasion or viral virulence existed (Tab. 5). When much more virulent virus is invaded, epidemic of human J. E. and protective immunity in aged people may be resulted as seen commonly in the epidemic in Honshu island.
So in one Hokkaido island, 4 areas, non-, sub-, temporary- epidemic and epidemic area co-exist. The borderlines of these 4 districts are determined on a map (Fig. 3). The borderline among non- and sub- epidemic area must be most significant.
Relationship with climatological data is discussed and the virus existence suspected to be controlled by some climatological conditions.

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