The search on the type distribution of coxsackie viruses among the group of children with undeterminable fever and their control groups was focussed on 37 isolations obtained from 359 fecal specimens in Okayama prefecture during 1954-55.
Our allowance to type these isolations were technically limited to A-1, 2, 3, 4, 5, 6, 8, 10, 19 and B-1, 2, 3, 4, 5. Two major strains designated as “Sakakiabra” which coupled with it isolations were apparently new to our scale, which were as well prevalent in two major grouoped child population and these both strains fulfilled the following criteria:
(a) production of paralysis of extremities in infant mice after an inoculation period of about 3 to 6 days,
(b) fatal termination of the disease in mice usually within 1 to 2 days from onset,
(c) reproduction of the disease in other infant mice on passage of bacteriologically sterile, infectious tissues, and,
(d) production of characteristic myositis in sick mice.
Furthermore 8 isolations were typed as A-2, 2 isolations as A-5 and 1 isolation as B-2.
The incidence of group infection of this fever and its sympto-matology gave rise to the affection by coxsackie viruses.

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As the preliminary study, growth characteristics of four viruses, i.e. influenza A (FMI), influenza B (Lee), Hemagglutinating virus of Japan (HVJ, Fushimi) and NewCastle disease virus (46-9674) have been examined in the tissue culture system using a piece of chorioallantoic membrane removed from 15-day old chick embryos as the host tissue. Among the four viruses tested, HVJ showed a characteristic growth curve when compared to the other three, i.e. long latent period for the cycle growth. The tissue culture system employed was also discussed with regard to its sensitivity of estimating alive virus particles, when compared to the egg infectivity titrations. In general, the former technic was more insensitive when compared to the latter in detecting particles and the difference lying between there two methods was most remarkable with HVJ.
In the culture system described above myxoviromycin, two different kinds of streptothricins, sinanomycin (netropsin) and G-72 (chartreucin) were tested for their activity to inhibit the virus growth. When the per cent inhibition was plotted on the ordinate against the log concentration of any drug on the abscissa, characteristic inhibition curve was obtained with each antibiotics. However, in order to inhibit the growth of four viruses, the concentration required with each antibiotics was almost in the same order. With higher inoculum size in the tissue culture system described above, higher concentration of myxoviromycin was necessary to inhibit the growth. However, the fact was not the case with the remaining antibiotics.

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The morphological characteristics of “Standard Passage” virus and “Undiluted Passage” virus prepared for electron microscopy by the usual air-drying and by freeze-drying were compared with each other, and the densities of these viruses were also compared. The results were obtained as follows:
1. SP virus isolated from allantoic fluids of infected eggs wee found to contain uniform particles of predominant by spherical shape with smooth surface, and as far as the appearence of particles was concerned, there was no great difference between an air-dried specimen and a frozen-dried specimen.
2. In contrast, UP virus obtained from allantoic fluid of infected eggs vas found to be pleomorphic and there were great differences between an air-dried specimen and a frozen-dried specimen. That is to say, in the air-dried preparation, the majority of the particles appeared flattened and doughnuts like, but in the frozen-dried preparation, the majority of the particles appeared appromimately spherical in form and flatted or doughnut like particles disappeared.
3. The density of the SF virus was estimated at 1.10 in blood albumin solution anti 1.23 in saline solution, meanwhile, the density of the UP virus was estimated at 1.07 in blood albumin solution and 1.16 in saline solution.
From these results, the existence of basic structural differences between the two types of virus was estimated.

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1. The nucleic acid contents of incomplete virus and complete virus (PR8 strain) were compared by U. V. absorption and color reaction measurements. (Orcinol-Hcl reaction and Diphenylamine reaction)
2. It was found, as judged by ultraviolet absorption measurement, that the complete virus contains about 3 times of ribonucleic acid compared with incomplete virus. (0.94%: 0.3%) Meanwhile, tests for pentose (Orcinol-Hcl reaction) indicated that the complete virus contains about 1.5 times of ribonucleic acid compared with incomplete virus. (2.7%:1.6%)
3. From these results, the reason of considerable disagreement existed in the literature concerning the nucleic acid content of influenza virus was discussed

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1) Using the photometric method, the incorporation of S antigen into incomplete virus and complete virus were compared with each other. At the same time, the amounts of S antigen formed in the infected tissues were also compared.
2) Decreasing the EID₅₀/HA ratios of virus suspensions, the amounts of S antigen that can be released by ether treatment from virus particles decreased, and the correlation between EID₅₀/HA ratio with CFS/CFV ratio of virus particles revealed that for each ten fold decrease in the former the CFS/CFV ratio decreases about two fold or so. The CFS/CFV ratios of each virus preparations were as follows:
SP virus: 1/1.5, UP₁ vircus: 1/2.5, UP₂ virus: 1/4.0, UP₃ virus: 1/8.0, UP₄ virus: 1/6.0, UP₅ virus: 1/6.0.
3) The infected allantoic membranes contained large quantities of free S antigen, and the free-CFS/CFV ratios in the infected allantoic membranes were obtained as follows: SP: 6.0, UP₁: 6.0, UP₂: 4.0, UP₃: 1.0, UP₄: 2.0, UP₅: 4.0.
4) The implications of these findings have been discussed.

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In this communication a report is on the cellular receptor for mouse encephalomyelitis virus (GDVII strain).
The results are summarized as follows.
(1) The CDVII viruses absorbed on lecithin particles were eluted in normal saline solution by heating at 37°C for 15minutes.
(2) The purified lecithin showed a high inhibiting titer enough to inhibit 1 hemagglutination unit of GDVII virus by 0.2γ/cc.
The inhibiting action of lecithin was not found on the hemagglutination of influenza virus (PR8), mouse encephalomyelitis virus (FA) and Japanese B encephalitis virus (Nakayama strain), but to some extent on the hemagglutination of lymphogranulomatosis inguinalis virus when human group 0 erythrocytes and calcium chloride solution were used.
(3) The purified lecithin was made from human erythrocytes, mouse brain and soy bean. The inhibiting action of the lecithin showed an almost similar activity, irrespective of materials.
(4) The lecithin inactivated only hemagglutination of the virus with no regard to the infectivity, and the virus reserved its infectivity even in the state of combining with the lecithin.
(5) The lecithin was made up of glycerin, unsaturated fatty acid, saturated fatty acid, phosphoric acid and choline. The inhibiting action of lecithin was inactivated by phospholipase B, C and D but therefore, not inactivated by phospholipase A. And the inhibiting action was observed in a constitution of glycerin, saturated fatty acid, phosphoric acid and choline, but unsaturated fatty acid seemed to be unessential component.
(6) The receptor of erythrocytes was inactivated by phospholipase A, B, C and D.
(7) The lecithin was separated from erythrocytes and mouse brain, and CDVII virus infected mouse brain contained the lecithin 16 times less than normal mouse brains.
All these findings would indicate that lecithin are surmised to be concerned in the cellular receptor for mouse encephalomyelitis virus (GDVII strain).

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In the previous report, an exploratory experiment to ascertain the presence or absence of similar modifying effect of bacteria or bacterial substances on the course of neurotropic virus infection to that of preumotropic virus in the natural state has been studied. The results obtained indicated that there are some bacteria having the inhibitory or stimulating effect on the virus infection.
The purpose of this paper is to report the mode and mechanism of the effect of E. coli which is selected among the bacteria having the inhibitory effect on the virus infection in an exporatory experiment. From the results obtained, it seems probable that the effect inhibits the connection with the virus and host cells from reasons of the following: the effect of E. coli is significant when the bacteria is given to mice two days before the virus inoculation; the most evident effect is obtained at the same route of virus and bacteria inoculation; the bacteria possess no direct virucidal activity; the anti-E. coli-serum is not related with the virucidal activity and virus neutralization; and both infection protecting activity and neutralizing antibody in the survival mice is not recognized. The effect of E. coli exists in the cells and thermostabile.

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The E. coli was selected among the bacteria having inhibitory effect described in the previous reports, and the experiments concerning the inhibitory effect of E. coli on the GDVII strain of mouse encephalomyeltisis virus (GDVII virus) were investigated in detail. The results obtained the following data: the administration with E. coli before virus inoculation was effective on the inhibition of the infection with the GDVII virus but when the E. coil was given to the mice following the virus infection the effect of the bacterium was not evident; it was most significant in the case of the same route of administration with the bacterium and the virus; the bacterium itself had not direct virucidal activity; the effect of the bacterium was not related with the antibody against this bacterium; and no evidence of inapparent infection in the survival mice was presented. Therefore it would be assumed that the inhibitory effect of E. coli on the virus infection might prevent the invasion or penetration of the virus into the susceptible cells.
The purpose of this paper is to report a part of the kinetics of the inhibitory effect of E. coli on the virus infection by using the Maitland' method in which the suckling mice brains were employed as the explants. The results obtained demonstrated that the inhibitory effects of E. coli on the virus were significant when the tissues were cultured by the fluid containing the bacteria before the virus inoculatio. However, at the prolonging the culture time, it was indicated that the difference of virus multiplication between the experimental and the control group was small in the beginning of the culture, as the culture was getting later, the difference also lager. Therefore no evidence was presented the mechanism was either prevension of the virus-cell combination or convarsion of an intracellular process for virus multiplication.

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During the epidemic period of Japanese encephalitis, most mammalians, especially horses and human beings, are infected with the virus, however, there are very few cases which reveal clinical symptoms. We are trying to determine the reason for this. In this report, the effect of sodium on the infection in mice is presented as one respect of the research concerning this problem. Diluent …… The diluent was beef broth (pH 7.4).
Virus …… The Nakayama strain of Japanese encephalitis virus was used in the form of centrifuged brain suspension from infected mice.
Mice …… The mice used were mostly of dd strain, their ancestors originating from Institute of Infectiond Disease, University of Tokyo. Adult mice were used when 5 or 6 weeks old.
Virus titrations …… Serial tenfold dilutions of infected materials were made in the diluent, and each dilution was inoculated (0.2ml) intraperitoneally or instilled intranasally into 5 or 10 adult mice immediately after the intracerebral injection (0.03ml) of Na-salt solutions or KCl solution, but control mice of the same age and same numbers were inoculated with the virus only by the same routes as the experimental mice. Mice were observed for 15 days and end-point then was calculated by the method of Reed and Muench. Deaths which occurred on the first two days were not included in calculations,
The results are as follows:
1. Six-week-old mice injected with 0.85% NaCl solution before intraperitoneal inoculation with the virus revealed an LD₅₀ of higher than 10^-4, whereas the LD₅₀ of control mice was lower than 10^-2 (no death occurred in the virus dilution at 10^-2).
2. Five-week-old mice injected with 0.85% NaCl solution before the intranasal inoculation with the virus revealed an LD₅₀ of 10^-3.74, whereas control mice revealed 10^-2.76.
In the case of intranasal inoculation, the difference in the LD₅₀ of experimental and control groups was smaller than that in the case of intraperitoneal inoculation.
3. The effect of NaCl solution was compared with that of KCl solution on the mortality of 5-week-old mice artificially infected with the virus in order to determine whether the effective factor was Na or Cl.
Mice injected with 0.85% or 0.085% NaCl solution before instillation with the virus revealed an LD₅₀ of 10^-3.37, whereas mice injected with 0.0085% NaCl solution, 1.0%, 0.85% and 0.5% KCl solutions, and aqua deat had LD₅₀s of 10^-2.62, 10^-2.62, 10^-2.32, 10^-2.50 and 10^-2.62 respectively, which was similar to the LD₅₀ of 10^-2.32 in control.
From these results, it is suggested that Na causes the enhancing effect on the mortality of mice, but KCl and aqua dent do not. It does not appear to be caused mechanically by the injection of the solution.
4. Other Na-salts had similar effects as follows:
Each group of 6-week-old mice injected with 0.1% NaHSO₄ rolution, 1.0% NaHCO₃ solution, 0.1% NaHCO₃ solution, 0.38% C₆H₅O₇Na₃ solution, 0.038% C₆H₅O₇ Na₃ solution and 0.85% NaCl solution res pectively, revealed the LD₅₀ of 10^-2.32, 10^-2.50, 10^-2.37, 10^-2.75, 10^-2.34, and 10^-2.34 respectively, whereas cntrol mice had less than 10^-2.00 (no death when inoculated with the virus at the dilution of 10^-2.00).

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During the period from January to May 1957, the distribution of neutralizing antibodies against Adenoviruses Type I to Type VI according to age was investigated by conducting tissue culture (Hela) on a total of 757 serum specimens taken from healthy inhabitants in Okayama District.
As a result of this investigation, the degree of prevalence of Adenovirus in Japan was clarified and compared with the distirbution of antibodies against Adenovirus in Washington Area for further study.
The result of this investigation was as follows.
1) The percentage of positive reaction of neutralizing antibodies according to age group in these 6 types showed almost the same changes.
Namely, it was found that the percentage of positive reaction was highest in the funicular blood and lowest 6 to 12 months after birth and increased rapidly during 3 to 5 years thereafter and slowly after 6 years.
This result suggests that each type of Adenovirus has prevailed very extensively in Okayama District.
2) This result was compared with the distribution of antibodies in Washington Area reported by Huebner. This comparison revealed higher rates in any type of Adenovirus in any age group in Okayama District than in Washington Area.
Namely, it was revealed that Adenoviruses were prevailing more extensively in Okayama District than in Washington Area.
3) In Okayama District, 50% of all infants ranging in age from immediately after birth to 12 months after birth were found to be infected with at least more than 2 types of viruses and adults have been usually infected with more than 4 or 5 types of viruses.
The percentage of infection in Okayama District was much higher than in Washington Area.

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During the period from 1955 to 1957, neutralizing serum reaction for Adenoviruses Type III and Type IV Was conducted on 36 cases of various infantile virus diseases observed in the Pediatrics Department, Okayama University.
As a result of this observation 5 cases of the 36 were found to be infection of these viruses.
1) One case of the 5 was an 11 month old boy. Clinically, he is a case of primary atypical pneumonia with no rise in cold haemoagglutinins. This case is infection with Adenovirus Type IV.
2) Two cases of the 5 were patients with febrile pharyngitis and conjunctivitis. They are cases of infection with Adenovirus Type III.
3) One case was a feverish patient without swelling of the parotid gland among a group of children attacked by mumps. This is a case of combined infection with Adenovirus Type III and mumps.
4) Another case was a 2 month old infant with roseola infantum. This is a case of infection with Adenovirus Type III.
Isolation of Adenoviruses was conducted on 47 infantile cases of various acute upper respiratory tract diseases using Hela cells from the throat washing. This isolation succeeded in one case. This was a patient with febrile pharyngitis and conjunctivities. Neutralization test revealed that this was infection with Adenovirus Type III.

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A simple technic has been developed for measuring PR8 influenza virus growth on preparations of chorioallantoic membranes together with the attached shell, removed from chicken embryos. 1) The maximum growth assayed by hemagglutinin was fairly constant. 2) The technic is simpler and less laborious than the usual method in which they used peeled chorioallantoic membranes from the shell. 3) During the continuous study for almost one year, there have been almost no chance of bacterial contamination. 4) Deviation of pH value from tube to tube after 24 hrs incubation was largely eliminated owing to the existence of shell.

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Freshly harvested allantoic fluid of PR8 (influenza) or Nagoya strain (HVJ) was centrifuged in a Spinco centrifuge. A condensed virus suspension with a titer of 20, 000 HA per ml was made from the deposit with saline. Sodium ethylmercurithiosalicylate (Merzonin, Takeda Co.) was added to the suspension in a final concentration of 1:10, 000. The virus particle was incubated at 37°C for 4 hours and kept in ice-box overnight and then washed twice in a Spinco with saline to remove Merzonin. Infectivity test of this Merzonin-virus with chick embryos indicated no viral propagation. Mice inoculated intranasally twice with a 0.1ml dose of Merzonin-virus in an interval of one hour died with extensive consolidation within four days. No infectivity of the consolidation lungs was confirmed with chick embryos. The lethal toxicity to mice of influenza virus or HVJ inoculated by intravenous or intracerebral route was lost by inactivation with Merzonin.
We would propose, therefore, that a viral component which can cause the formation of consolidation is to be named “consolidation factor”. The mechanism of production of pulmonary consolidation was discussed in connection with Reilly's phenomenon.
Accelerated death of mice previously sensitized subcutaneously or intranasally with Merzoninvirus of influenza occured only when the Merzonin-virus was given intranasally, but dit not by other route. As the mice showed intensive bronchiospasm at autopsy, it seems to be that the acute death was the result of an allergic reaction following the attachment of virus particle to the sensitized respiratory epithelium.
Relationship between the consolidation factor and the allergic antigen attributed to the particle was discussed.

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In pursuing the growth of influenza virus in animals, particularly by means of hemagglutinin, or in performing the tissue culture experiment with influenza virus by means of infectivities again in tissue culture or of hemagglutinins, it was thought expedient to attempt to characterize the nature of both hemagglutinin inhibitors and growth inhibitory substances, detectable in animal sera or organs. The way how to destroy these inhibitors was also the matter of importance in performing these kinds of works. The study here described, summarized the results obtained along these lines of experiments.
In the first series of experiments, non-specific hemagglutination inhitors found in biological specimens have been divided into four groups. The first one was the so-called β-inhibitor and was contained in bovine serum. The representative of the fourth group was the guinea pig serum and its inhibitory action was destroyed with KIO₄ but not with purified RDE. The second and the third groups were the already known α-inhibitors, but were differentiated each other by their specific inhibitory spectrum.
In the second set of experiments, the specimens tested above were examined for their growth inhibitory activity on the PR8 and CAM virus in Maitland's type tissue cultures. Chicken chorioallantoic membrne was used as the host tissue. Except the horse serum, all sera tested contained this kind of activity against these 2 viruses. Though the elucidation of this activity was not accomplished here, further development of this kind of study seems to be promissing.
Finally, the third approach involved here, was to apply the results obtained above in analyzing the distribution of these inhibitors against the PR8 virus particularly in mice. Lung, kidney and spleen contained fairly large amount of hemagglutinin inhibitors, which were susceptible to the action of RDE. Growth inhibitory substances were found only in serum and liver of mice and the latter was studied somewhat in detail with regard to its site of action. The inhibitory action, however, was not so high and the participation of this inhibitor in experimental infections were not elucidated so far.

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According to a number of investigations published up to date, fresh active NDV had to be inoculated in a considerable amount to cause toxic effect or to produce a lung consolidation, and the inactivated virus was considered to be completely ineffective. In the present study, it was found that mice inoculated intranasally died of lung consolidation even when NDV had been inactivated previously by sodium ethylmercurithiosalicylate. Basing on this and other results obtained, a conclusion was drawn that infectivity, hemagglutination and activity to produce lung consolidation are respectively independent attributes of NDV, in which that concerned with lung consolidation was named “consolidation factor”.
Intravenous injection of fresh NDV caused a marked intestinal hemorrhage of mice, which was not produced with inactivated virus. Lung consolidation and intestinal hemorrhage were strikingly protected by an appropriate use of antonomic blocking agents such as tetraethylammonium bromide or chlorpromazine. From these facts it has been postulated that the pathological changes produced by NDV are results of an irritation in vegetative nervous system which may be caused by the components of NDV. It seems to be that in lung inoculated with NDV the consolidation factor may affect the vegetative nerve endings of respiratory tissues and its excess excitation results the consolidated lesions.

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1) Ackermann and Kurz's report about the growth of Influenza virus in Ehrlich ascites tumor was confirmed by our stock strain of Ehrlich ascites tumor and Ackermann's strain of Influenza virus. In addition the process of oncolysis was examined cytochemically and cytologically. It was also noted that the ratio of infectivity to hemagglutination titer of infected ascites fluid was low.
2) A new host virus system, AsWS influenza virus and Yoshida ascites tumor system was reported. In this system, the growth pattern of the virus was different from that in the system of Ehrlich ascites tumor in two points, a) hemagglutination was observed only in the emulsion of cell sediment of infected ascites fluid and not in supernatant., b) growth curve showed a peak at 2 or 3 days after infection.
3) The process of oncolysis of Yoshida ascites hepatoma by the virus was examined cytologically and cytochemically.
4) Relationship between the capacity to support viral propagation and canceriyation of liver cells by DAB was discussed.
The anthers wish to express their appreciationto Dr. K. Ogasawara and Dr. S. Hibino for their helpful criticism.

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