1. Attempts were made to isolate mumps virus by means of amniotic inoculation of developing hen's eggs and a virus was isolated which was antigenically identified as same as the Enders strain of mumps virus.
2. Investigations were made in a certain fairly isolated community to obtain an information of the distribution of the antibodies (hemagglutination-inhibition antibody and complement-fixation antibodies) against mumps virus. (Refer to page 1-6.)

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The Hawaiian strain of dengue virus was purified from developing chick embryos and suckling mice brains with protamine clarification and ultracentrifugation. Twenty per cent solution of developing chick embryos infected was made with pH 8.5 Ringer solution and extracted in the cold for 3 days to disintegrate normal component. However, in the case of mice brains ten per cent suspension was prepared with double distilled water and immediately subjected to the treatment with protamine sulfate (salmine). Dengue virus remained in protamine supernate as expected because of its small size, and was centrifuged at high speed, approximately 85, 000g for 60 minutes.
Log of LD 50/g nitrogen of a final purified material, suspension of pellets in buffered saline, from developing chick embryos was 11.4 while that from suckling mice brains 13.3. (Refer to paege. 6-11)
Acknowledgement
This study was carried out at the Department of Bacteriology and Parasitology, University of Chicago in 1951 under the aid of Mc Cormick Fund. The author wishes to thank Dr. Robert Rustigian for his helpful advice and greatest support.

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Experiments were carried out with Escherichia coli (Strain B) bacteriophage, T₂ and involved multiple infection of the bacterial host.
The infected bacilli were treated after 5 minute-, 10 minute-, or 18 minute infection, respectively. Sectioning technique of bacteria deviced by myself (at the Dept. of Infectious Diseases, School of Medicine, University of California in 1950) will be outlined as follows:
At well-defined times during the latent period, the infected bacterial cells were fixed in 0.5% osmic acid for 10 minutes. After washing and dehydration through alcohol series, bacterial pellets were embedded in methacrylate monomer, and one-tenth micron sections were cut with the Minot Ultramicrotome. Electron microscopy of sections of the infected bacteria was done with the J. E. M. type III electron microscope.
The results obtained may be summerized as follows:
a. Phage particles could not be observed in any section of the bacteria of 5 minute infection, while protoplasm often appeared in swelling in 2 or 3 parts as those shown in Fig. 1. (See page 13.) and it was noticeable that the bacilli occasionally showed a forky developing form.
b. After about half of the latent period (10 minute infection), characteristic, rather dense intracellular particles or areas were observed which seem likely to mature to morphologically perfect phage particles by the time of phage lysis. (See Fig. 2, page 13.)
c. In a sample of 18 minute infection, a large number of newly formed (replicated) T₂ phage could be clearly seen in sectioned cells. The size of every phage measured 60×80 mili-micron, i.e., the same size as mature phage. Definitely it had a limiting membrane completely corresponding to mature phages. (See Fig. 3, page 14.)
Some of the cells of 18 minute infection became considerably fragile and entangled delicate fibers present in the protoplasm could be seen.
In 1951 I have tried phage studies of the same line as the present work under Dr. M. Delbrück's guidance at the California Institute of Technology.

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In the study of the initial stages of phage infection to its susceptible host cell, it is of interest to determine the character of the binding force which acts highly type specifically between host and virus, in other words, to decide whether it is merely ionic force or it contains some enzymatic processes.
From this point of view, the influence of temperature on adsorption rate of phage to host cell was investigated.
This report represents some adsorption experiments with two different host-virus-systems, one is temperature dependent and the other is unaffected by temperature in character with respect to attachment affinity between host and virus, and about the character of affinity force is discussed.
Experiments with P6r phage and its host cell E. coli E6 showed that adsorption velocity became smaller as the temperature lowered but it is chiefly due to increasing of the viscosity of the medium and adsorption efficiency was kept constantly in the neighborhood of 100 per cent, telling the binding force acted between these colloidal particles was unaffected by the temperature, in the range between 37°C and 0°C, and at the same time, it demonstrates that diffusion theory holds for this phénomenon with well agreement.
On the other hand, experiment with T4r phage and its host E. coli B revealed that there existed the distribution of attachment activity among phage particles related to temperature and, as the result of this, amount of adsorbed phage particles was markedly influenced by the temperature. (See page 16-23.)

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L'immunsérum contre un virus empecherait-il l'adsorption du virus sur la cellule hôte? Le virus neutralisé par l'immunsérum ne s'adsorberait-il pas sur la cellule hôte?
Dans les présentes expériences, les bacilles ont été laissés en contact avec le lysat bactériophagique incomplètement neutralisé. On a examiné si, chez les bacilles ainsi traités, se produisent les phénomènes suivants: 1° l'interférence avec les phages homologues, 2° la réactivation de phage (“multiplicity reactivation”), 3° la suppression de la formation de colonie et 4° la lyse sans croissance de phage (“lysis from without”)
Nous n'avons constaté aucun de ces phénomènes. Il reste obscur si le phage neutralisé ne s'attache point à la bactérie hôte ou si le phage neutralisé s'adsorbe sur le bacille sans provoquer aucun des phénomènes ci-dessus mentionnés.
C'est ainsi que nous ne sommes pas arrivé à mettre en évidence l'adsorption du phage neutralisé sur la bactérie hôte. (See page 27-24.)

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Within the past two years, since the autumn of 1950, scattered outbreaks of unknown etiologic polyradiculoneuritis, “Landry-Guillain-Barré's syndrome” occurred in these parts bordering the Inland Sea of Japan.
I. During the summer of 1952, the neutralizing antibodies of 9 sera of patients with Landry-Guillain-Barré's syndrome and 10 sera of poliomyelitis patients (spinal form) were examined for the Lansing strain of poliomyelitis virus. Five sera of Landry-Guillain-Barré's syndrome were positive; 3 sera of them were equivocal; only one serum was negative. But in the cases of poliomyelitis only 2 sera were positive; all other sera were negative.
On the other hand, the average age of Landry-Guillain-Barré's patients was 6 years and that of poliomyelitis patients was 2 years.
Serum antibodies capable of neutralizing the Lansing type become more frequent with increasing age during childhood, and therefore, our results seem to be insufficient to show what relationship this type of virus bears to the etiology of Landry-Guillain-Barré's syndrome.
II. During the summer of 1952, the hemagglutination inhibition antibodies of 8 Landry-Guillain-Barré sera and 5 poliomyelitis sera were examined on the hemagglutination of O-type human erythrocytes caused by New Castle disease virus.
But in all tested sera hemagglutination inhibition antibodies were negative.
Therefore it seems that there is no etiologic relationship between Landry-Guillain-Barré's syndrome and New Castle disease virus. (See page 27-31.)

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Within the past two years, since the autumun of 1950, scattered outbreaks of unknown etiologic polyradiculoneuritis, “Landry-Guillain-Barré's syndrome” occurred in these parts bordering the Inland Sea of Japan.
During these time 15 (23%) of 65 patients who were admitted to our children's clinic hospital died with bulbar paralysis.
During the summer and autumn of 1952, we discovered the virus pathogenic for suckling mice in feces obtained, during the acute stage of illness, from 2 children out of 12 patients, with the methods of Beeman et al. (1952) for the isolation of Coxsackie viruses.
The first strain was isolated from patient S. Dohi, a 5-year-old boy (Dohi strain) and the second was isolated from H. Okumoto, a 4-year-old girl (Okumoto strain), (See page 32-39.)
The properties of our two strains of virus and the results of neutralization tests were as follows.
1. The oldest mice susceptible to Dohi strain were 14 days old, and the oldest susceptible to Okumoto strain were 16 days old.
2. The LD₅₀ titre, log of dilution, of the infected mouse muscle of Dohi strain was 6.00, and that of Okumoto strain was 7.00. The LD₅₀ titre of the infected mouse brain of Dohi strain was 4.00, and that of Okumoto strain was 6.00.
3. The neutralization index log of patient S. Dohi serum collected on 27th day after the onset of illness was 5.50 for Dohi strain, and on the 191st day it was decreased 50 fold.
The neutralization index log of the patient H. Okumoto serum collected on the 3rd day was 1.23 for the Okumoto strain, Unfortunately, because H. Okumoto died on the next day, the convalescent serum could not be tested.

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Morphology, pathogenecity and serological activity of purified TMV have been studied under treatments with temperature, sonic waves, formalin, extract from the leaves of Phytolacca americana L. and “bukulin” extracted from Pachyma hoelen Rumph.
1. In experiments with varying temperature no change was observed at 75°C. At, 80°C or a higher temperature the number of about 280mμ long rods was considerably reduced and the number of short particles accordingly increased. The number of the latter became very large at a temperature of 90°C or more. With the increase of the number of small particles the pathogenecity was decreasing, while, on the other hand, the serological activity was growing.
2. In experiments with sonic waves the longer the treatment lasted the more reduced became the length of the rods; they did not reach the size mentioned above. The pathogenecity was reduced and the serological activity increased.
3. In experiments with temperature and sonic waves the length of the shortest particles was about 25mμ or still less, and their number was increasing in advanced treatment stages.
4. In experiments with temperature the short particles, enlarged in size, showed more or less irregular hexagonal contours. Sporadically they were seen standing upright; the same phenomenon could be observed under treatment with sonic waves.
5. In experiments with sonic waves though the rods underwent segmentation, often the particles did not disperse. It is supposed that they were kept together by some substance. (See Fig. 1-Fig. 12, page43-48.)

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I have studied the interference phenomena between Influenza virus and several viruses including Japanese B Encephalitis in chick embryo system. When several strains of Influenza virus were inoculated into the chorio-allantoic cavity of an embryonated egg, inoculated 72 hours previously with Japanese B Encephalitis virus, the Influenza virus was unable to propagate. The excluding ability of Japanese B Encephalitis virus against Influenza virus depends upon its virulence; also the interference phenomena of Japanese B Encephalitis is stronger when inoculated into the yolk sac than when into the chorioallantoic cavity.
From the above experiments, with Influenza virus as the indicator the degree of propagation of the Japanese B Encephalitis virus in hatching chick embryo can be detected.

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The author observed the lysis of Escherichia coli with phage by means of adding broth culture (0, 1ml) into phage of various concentration (10ml) and comparing the turbidity with the standard. As lysis was observable with Sh. Komagome A phage and Escherichia coli, the observation was chiefly done with this case. The results obtained are as follows.
1) With Escherichia coli, lysis was often unobservable. When the lysis was observable, it was still seen, even if the number of cells or the ratio of phage particles to bacteria was changed widely at the time of infection.
2) Shaking of the solution had a strong influence upon the turbidity curves.
3) The lysis curves changed according to the concentration of the added organisms.
4) There was no great difference between the bacteria that had been cultivated under the short-time aeration and the one that had been cultivated for 18 hours.
5) When the author operated Sh. komagome A phage on Sh. komagome A, Sh. saigon and E. coil, each of the three showed about the same sensitivity against this phage. However, there were quite large differences in their lysis curves. (See page 54-59.)

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If the vaccinator assumes that all of the people who revealed immune reaction have enough immunity to smallpox, he may be misled about the state of resistance, unless he used potent vaccine and payed much attention to the local resistance of the site of inoculation, he might have inoculated on the most resistant area. There are evidences that at the sites of previous vaccination a local immunity may develop.
In order to investigate the relationship between the site of inoculation and the rate of successful results, we divided 114 students in two groups and vaccinated the one on the left forearm and the other on the left upper arm. Table 1 shows the rate of successful results of the both groups. The rate of successful responses of the former group is 30-40% higher than that of the latter.
Then, 119 children received two scarificatioris on the left forearm and two on the upper arm. As seen in Table 2, the rate of successful results on forearm is much higher than that of upper arm. Table 3 shows hat out of 30 children reacted immune on the upperarm, 23 received successful vaccination on the forearm. The contrary. example never existed. So, many of the immune reactors on the upper arm are, so to speak, pseudo-immune reactors and should not be considered to have enough immunity.
The rate of successful results of the forearm vaccination with potent vaccine was more than 80%.
Today, as smallpox has been successfully controlled, the medical profession has become so neglectful of vaccination that many of the people have not enough immunity to the disease. There is a danger of spread of smallpox in such a state of mass immunity, if the disease is introduced in the country.
Summary. It is preferable to avoid the sites of previous vaccination, for there are evidences of local immunity as we have shown in this paper. Forearm vaccination with potent vaccine is recommended as the most effective mass immunization against smallpox. (See page 60-64.)

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Three strains of the rickettsiae of Tsutsugamushi disease, Pescadores, Fuji and Niigata strains, were inoculated intracerebrally to mice and the propagation of them in the brain were quantitatively examined and pursued. The rickettsiae propagate there drawing almost a parabolic line and attain its peak approximately ten days after the inoculation. The maximum LD₅₀ titer attained in the brain is almost as high as that which can be attained in the liver.
Serial brain to brain passage in mice with the interval of seven to ten days was made for forty generations without loss of virulence or remarkable change of properties.
The infected brains were made to microscopic preparations and the rickettsiae in them were examined with the Giemsa-staining. Meninges, ependhyms or choroid plexus are the predilections sites of the rickettsiae for their propagation, but in nerve cells or glia cells no rickettsiae can be found at all. Namely it is observed that also in the brain the rickettsiae attack and propagate in principally the cells of mesodermal origin as already known in other susceptible organs.
In the course of intraperitoneal passage of the rickettsiae in mice the irresistible contamination by intestinal bacteria proper to mice is sometimes, especially in summer season, inevitable. In consideration with the above fact, the intracerebral passage, which is much simpler and easier in manipulation than the former and absolutely free from contaminations, except those dueing to technical mistake, is certainly a preferable method for the routine preservation of the Tsutsugamushi disease rickettsiae.

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