The mechanisms of multiplication of virus was studied by biological and pathological analysis of the process of multiplication inoculated virus on embryonated eggs in which metabolic changes were aroused in the cells by the x-ray irradiation.
1) First of all, the tolerance of embryonated eggs against x-ray irradiation was studied. When dose of 1000r was given, 80% of embryonated eggs were succumbed 12 hours after the time of irradiation and death rate reached 100% 24th hours after the time of irradiation.
When 800r was given to the eggs, death rate reached 100% 24th hours after the time of irradiation. Irradiation of 100r killed 30-50% at the 24th hours after the time of irradiation, while irradiation of less than 500r brought no death among them.
2) The significant decreases of weight of embryonate foetus were observed 62 hours after the time of irradiation of 100r and 500r.
3) A certain amount of virus was inoculated on embryonated eggs at 90th min. 14th hour and 26th hour after 600r was given and hemagglutinin titers were measured and compared. Distinct differences were noted between them. The result showed that the multiplication of virus was accelerated in 90th min. and 14th hour group and inhibited in 26th hour group.
4) The effect of x-irradiation to allantois were discussed.

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The mode of multiplication of influenza virus were studied by measuring hemagglutinin titers when 600r was irradiated 60min. and 180th min. after they were inoculated in allantoic cavities.
The result revealed that their growth was distinctly accelerated than in control groups. However, titers obtained at 6th hours and a half and 8th hours after the inoculation showed that viral growth was inhibited at these both stages where complete virus was increasing.
The invasion and damage of allantoic membrane occured about two hours earlier in the group which was given 600r 3 hours after the time of inoculation than in the group which was not irradiated.

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The process of viral synthesis in the allantoic fluid and chorioallantoic-membrane was studied when various doses of x-ray irradiation was given 3 hours after the time of inoculation influenza virus on embryonated eggs. At the same time, chorio-allantoic membrane was histologicaly studied and compared.
1) In the group which was given 50r and 10r, viral synthesis was markedly inhibited at the initial stage of virus multiplication, however the production of complete virus particles in the later stage was as good as observed in control groups.
2) After irradiation with various dosages of x-ray, the hemagglutinin titer varied almost proportionally with the infectioe titer.
3) There is no significant difference in amount of DNA between the irradiation and control groups, but as for the RNA changes, it varies proportionally with the viral growth curve.
4) During 8-16 hours after the time of inoculation when the growth of virus is most marked the distinct pathological changes were observed in the ecto-and mesodermas, while slight changes were noted in the endoderma.

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In the course of the study on the interference of vaccinia virus inactivated by ultraviolet irradiation with vaccinia infection, an inhibitory factor separable from virus particles was found in the skin and testicle of the rabbit infected with vaccinia virus. This factor was not detected in the chorio-allantoic membrane infected with the virus in spite of abundant multiplication of virus.
This factor exerted inhibitory effect on dermal infection of rabbits with vaccinia virus when active virus was inoculated in the site of the skin pretreated with this factor.
Marked inhibition was observed when this factor was injected one or two days before active virus and little, if any, effect is noticeable when this factor was administered immediately before or as early as 14 days before active virus. These findings suggest that this phenomenon has nothing to do with neutralizing antibodies.
The activity of the factor was not affected by heating at 56°C for 30 minutes nor by ultraviolet irradiation enough to destroy the interfering activity of virus particles. The factor remained in the supernatant after centrifugation at 10⁵×g for 2 hours, was readily filtered through Seitz EK pads, and not dialisable through cellophane bags.

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Virus growth in several organs of mice after intranasal, intracerebral and intraperitoneal inoculations of influenza virus, PR 8, was first studied.
Even the inoculum was given from parenteral routes, the highest growth was obtained in lung among the organs tested. The mouse here used was three-weeks-old one and the virus titer in lung after intraperitoneal and intracerebral inoculations of large dosis of virus was just comparable to that obtained after inhalation of the small inoculum. In spite of such good growth, mice did not succumb after parenteral injections and sudden fall of virus titer in the lung was remarkable on the fifth day. Early and high antibody rise at these occasions was correlated to this sudden fall of virus titer.
The significance of hemagglutinins with low infectivity detectable in kidney and liver particularly at the time of parenteral inoculations was discussed.

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1) The results of mouse neutralisation tests with intraspinal inoculation technic were compared with those obtained by HeLa cell tissue culture neutralisation method, using three_ types of poliomyelitis virus vs. diluted sera (1:10) from 12 typical cases of paralytic poliomyelitis. Only one serum specimen gave a clear cut positive by tissue culture neutralisation test, but negative results by mouse neutralisation test, and the remainder showed complete agreement between the results of both methods.
This preliminary study indicated that the neutralisation tests in mice with mouse-adapted polioviruses were utilized effectivly in circumstances which tissue culture neutralisation test could not be done.
2) In 1955, 111 sera were collected from inhabitants of Okayama area. The percentage positive of neutralizing antibodies examined in mice with these diluted sera (1:10) against 100 PD₅₀ of type I and II, and 20 PD₅₀ of type III were followed: in placenta cord serum, against type I 83.3%, against type II 66.7%, but type III 33.3%; thereafter these percentage decrease rapidly, reached the lowest level in 6 months to 1 year group. Then the positive percentage against type I and II sharply rose up from 2-4 years group and reached the adult level in 10-14 years group against type I and in 5-9 years group against type II. But during 2-19 years old, the positive percentage against type III were still remained about 20-29%. These pattern of antibody-level illustrated that the distribution of type I and II of poliomyelitis virus in Okayama were resemble to those of Tokyo and Osaka, but the one of type III showed endemic nature and its infections seemed not to be prevalent in our area.

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It is reported that mephenesinpropyleneglycol with tetracycline is clinically useful for the treatment of Japanese encephalitis. So we studied on virucidal effect of mephenesin against Japanese encephalitis virus by mice-inoculation test. Challenge to mice with Japanese encephalitis virus was tried with intraperitoneal inoculation and the mice were observed for 14 days after virus challenge.
10^-1 of Japanese encephalitis virus infected mouse brain and mephenesinpropyleneglycol was injected intraperitoneally into mice. Mortality of mice injected with 1.25mg. of mephenesin was beneath of 60%, but that of mice injected with 0.62mg. of mephenesin was 80%.
Mice inoculated with Japanese encephalitis virus were injected with 0.125mg. of mephenesin at 1, 6, 12, 24, 48 or 72 hours after virus challenge, but mortality of the mice was about 80%. 1, 7, 13, 19, 25, 36, 48 and 72 hours after challenge with Japanese encephalitis virus, mice were injected with 0.025mg. or 0.012mg. of mephenesin at each time, but mortality of the mice were high.
In the similar experiments with mephenesin and chloramphenicol or with mephenesin and tetracycline, we did not recognize virucidal effect of mephenesin against Japanese encephalitis virus.
Our experiments show that the drug has weak ability of inactivation of Japanese encephalitis virus only in vitro, but that in vivo has no effect.

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By the hemagglutinaton-inhibition tests with human convalecent sera it was found that both P and Q phase viruses were found in strains of Asian influenza virus.
These viruses appeared to be similar to one another antigenically but differed in their avidity for antibody.
In the course of these studies four main differences were found between P and Q strains; avidity, for antibody, sensitivity to nonspecific inhibitors, heat resistance of hemagglutionin, and agglutinability of cow red cells.
Q strain of Asian virus were not sensitive to the unstrapped 2-inhibitor and showed heat resistance of hemagglutinin. P strains of Asian virus and all strains of influenza A including swine influenza failed to show the agglutination of cow cells, but Q viruses agglutinated cow cells. The agglutination produced by Q viruses was similar to that of Lee strain of influenza B. After intranasal passages of Q virus in mice or treatment with periodate, a change of Q to P phase could be demonstrated.
The P variant induced in such a way reacted to efficient titer with human convalecent sera and with non-specific inhibitors, and lacked the heat resistance and agglutinabilty of cow cells.
These data suggested that reactive group of Q strain was subsurface in virus particle.

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Serums of three normal humans which had the activity of destroying HeLa cells were accidentally found when we were cultivating the cells using serums purchased from a blood bank in Tokyo. Subsequently three viral strains were isolated from these serums. They were found to be strains of a single virus which has been designated as the HSO virus (Human Serum Orphan). The virus has the following properties:
1) It is filtrable through Seitz EK, Chamberland L₃ and Berkefeld N and readily sedimented by centrifugation at 80, 000×g for 60 minutes.
2) It is rapidly inactivated at 56°C in 12 minutes, and gradually at 37°C. It is very stable at -20°C.
3) It is not inactivated by ethyl ether.
4) It well multiplies with cytopathogenic effects in cultures of HeLa cells or cells of skinmuscle tissues of the human embryo, but shows no evidence of multiplication and cytopathogenesis in cultures of monkey kidney cells. Cytopathogenesis is shown in cultures of H946 strain of human bone-marrow cells, Chang's strain of human liver cells or Henle's strain of human intestinal cells, but not in cell cultures of chick embryo tissues.
5) It has no pathogenicity to mice, suckling and adult, guinea pigs, rabbits or developing chick embryos. The Japanese monkey (M. fuscata) seems to have low susceptibility to the virus, showing leucopenia upon infection.
6) It is not neutralized by antiserums against the viruses of influenza (PR8, FM1, Lee), HVJ, Japanese encephalitis, herpes simplex, vaccinia and Rift Valley fever. No cross-neutralization occurs between HSO virus and the viruses of polio Types 1, 2 and 3, ECHO Types 1-7, 9 and 12, Coxsackie A Types 11, 13, 15 and 18, and adeno Types 1-14. The three strains of HSO virus are not distinguishable from each other by neutralization test.
After due discussion of the differentiation from the known viruses it was concluded that HSO virus is a new virus.
Neutralizing antibodies for HSO virus were found in 14.5 per cent (14/92) of adults and in 22.7 per cent (5/22) of children, 8 to 10 years of age, a fact indicating that the virus is disseminated among human beings. However, no data have been yet obtained in relation to clinical manifestations in human beings which might be produced by HSO virus infections.

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In order to know complement fixing (CF) antibody response against each of 3 type polioviruses following injection with Salk's type vaccine made in U.S.A. in infants under 3 years old and school children aged 6-12 years in Japan during the period covering from May to June, 1956. Another group of 20 infants served as control.
Out of 130 infants to be vaccinated 107 were proven to be triple negative in CF test and were devided into 3 groups, A (79 infants), B (15) and C (13). Groups A and B were injected twice and three times subcutaneously with 1.0ml dosis each, 7 days apart respectively. Group C were inoculated twice intradermally with 0.1ml dosis each, 7 days apart. The school children were vaccinated according to group A and the control group were twice injected with 1.0ml dosis of saline each, 7 days apart. Beside such a primary immunization, another shot was given as a booster immunization 7 months after the initial shot of the primary immunization. Serum was collected from each vaccinated individual 3 times, before vaccination, and each two weeks after the last shot of the primary and booster immunizations. And one year thereafter blood samples were collected from vaccinated infants as possible as we could.
CFT was carried out according to the Black and Melnick's method which was little modified by us. On the other hand, the vaccination was done in 160 infants under 3 years old during the period from September to December, 1957, in order to confirm the results obtained after vaccination by both subcutaneous and intradermal methods respectively in 1956.
The positive change rates against each type of polioviruses in 79 triple negative infants among group A were given as follows: 32% against type I, 27% against type II, 18% against type III and 25% on average after the primary immunization, and 52% against type I, 64% against type II and 46% against type III and 54% on average after the booster immunization, and 32% against type I, 24.% against type II and 16% against type III and 24% on average one year after the initial shot. It is worthy to note that there were no significant differences among the positive change rates after the primary immunization in 15 triple negative infants among group B and 13 negative infants among group C and that no marked difference in the positive change rates was observed between group A and B.
If the positive change rates in 3 groups, A, B and C are criticized not quantitatively but qualitatively, that is, in the ratio of children with titer more than 1:16 to all positive ones, no marked difference was recognized in ratios between groups A and B but the ratio in group C was found lower than those in groups A and B. Consequently it seems likely that 3 shots are not necessary for the primary immunization and that the subcutaneous injection of the vaccine may be preferable for the vaccination.
To observe the immune response after the vaccination from the standpoint of each age group, 30% against type I, 15% against type II, 5% against type III and 17% on average after the primary immunization, 32% against type I, 42% against type II, 42% against type III and 39% on average after the booster immunization were given under 1 year old, and 31% against type I, 23% against type II, 20% against type III and 25% on average after the primary immunization, and 67% against type I, 74% against type II, 59% against type III and 67% on average after the booster immunization in 1 year old. Now it has been made clear that the immune response after vaccination would be lower in triple negative infants under 1 year old than 1 year old. The positive change rates after the booster immunization in triple negative children aged 1 to 3 years were almost the same to 50% after the primary immunization in 14 triple negative school children. In general speaking of the ratio of number of positive children with titer more than 1:16 to all positive children in age groups, the ratio in infants under 1 year old

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In detecting antibodies against influenza virus, hemagglutination inhibition titration still remains as a most handy and useful procedure. However, as many one noticed at the outbreak of Asian flu, precautions must be taken in order to destroy non-specific inhibitors contained in the serum specimens, particularly when Asian P virus was used as antigen. The urgent problem at the time of outbreak was dissolved firstly by using KIO₄ to remove these inhibitors. However, some destruction of antibody was recognized with this technique. The work here presented was carried out to establish_ the standard procedure to destroy all kinds of non-specific inhibitors but not antibodies.
The result pointed out that when a large amount of cholera filtrate (No 558) was added to the specimen and left over night at 37°C, all inhibitors were removed as was the case with KIO₄ treatment. Moreover, it was concluded that the cholera filtrate is more suitable for general purposes, i.e. to destroy inhibitors of the serum or organs of experimental animals except serum of guinea pig.
As already noticed, inhibitors against Asian P virus detectable in human serum (tentatively called as α′-inhibitor) was resistant to RDE action, but susceptible to the action of cholera filtrate. Thus the difference between cholera filtrate and RDE was studied further, and the term α′-enzyme was proposed to the particular agent which destroys α′-inhibitor or inhibitors. The kinetic study revealed difference between α- and α′-enzyme. The necessity of Ca ion as the co-factor was also different. However, heat lablity of α′-enzyme was most remarkable in differentiating it from α-enzyme. Electrophoretic mobility was also somewhat different.
Production conditions and standardization procedure of the cholera filtrate were also described. When the experimental conditions here described was followed carefully, the production of RDE (α-enzyme), α′-enzyme and β-enzyme of the filtrate was constant during the experimental period of 6 months.

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This paper deals with some results in the observations on the metabolisms of green rice leafhopper, Nephotettix bipunctatus cincticeps UHLER, when it becomes viruliferous with the rice stunt virus, viz. Oryza virus 1.
(1) The values of the oxygen consumption (Qo 2) and the respiratory quotient (RQ) of the viruliferous leafhopper are larger than those of the nonviruliferous one. The respiration of the viruliferous individual seems to become more vigorous as affected with the virus examined (Table 1 and Fig. 1).
(2) The oxidative phosphorylation or P-O ratio shows larger value in the viruliferous than in the healthy leafhopper. It appears to be more vivid in the diseased insect to esterify inorganic phosphate acompanied with oxygen consumed (Table 2).
(3) The succinic dehydrogenase activity is hardly influenced in the insect vectors as affected with the virus examined (Fig. 2).
(4) The phosphatase activity becomes slightly stronger in the viruliferous leafhopper than in the nonviruliferous one (Table 3).
(5) The phosphates fractionated in the leafhopper are shown in Table 4 and Figure 3. The amount of every phosphate is larger in viruliferous than in nonviruliferous individual. Especially the nucleic acid phosphate increases remarkably in weight as infected to the virus.
(6) In the ultraviolet absorption spectra of nucleic acids of the leafhopper, the peak in the optical density of the acid is found on 260mμ in nonviruliferous and on 255mμ in viruliferous leafhopper (Fig. 4).
(7) The paper electrophoresis of the nucleo-protein in the leafhopper presents 3 fractions (I, II and III) both in the healthy and diseased protein. But the protein of the viruliferous insect decreases in the volume of the fraction I and II, excepting the fraction III (Fig. 5 and 6).
From the results obtained, it is to be concluded that the respiration and protein metabolism of the leafhopper examined become rather unsound when it is viruliferous with the rice stunt virus.

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The interference o f the neurotropic strain with the pantropic strain of Rift Valley fever virus was studied in mice.
1. Neurotropic virus, when inoculated intraperitoneally, interfered with intraperitoneal infection of pantropic virus and a decrease in mortality rate and a prolongation of the death-time were observed.
2. Neurotropic virus of 10^7-8×LD₅₀ was necessary to interfere with pantropic virus of 10³×LD₅₀.
3. When pantropic virus was inoculated 24 hours before neurotropic virus, the interference did not occur. Pantropic virus inoculated simultaneously with or immediately after neurotropic virus was interfered.
When pantropic virus was inoculated thereafter, the interference gradually changed to the immunity without any definite border-line separating them.
4. Neurotropic virus inoculated intraperitoneally interfered with pantropic virus inoculated intraperitoneally, subcutaneously, intravenously or intracerebrally. When inoculated subcutaneously, neurotropic virus slightly interfered with pantropic virus inoculated subcutaneously at a different site, but not with pantropic virus inoculated intraperitoneally, intravenously or intracerebrally.
5. When the interference occured, the multiplication of pantropic virus was strongly inhibited. But effect on the multiplication of neurotropic virus could not be determined. When the multiplication of pantropic virus was sufficiently inhibited, some of the mice died of infection of the brain with neurotropic virus and the remaining mice survived, as observed in mice inoculated with neurotropic virus alone. Or, some animal died of infection of the brain with pantropic virus. When the multiplication of pantropic virus was not sufficiently inhibited, the animal died of systemic infection with this virus, but the deathtime was somewhat prolonged.
6. Emulsions of normal mouse brains had not interfering activity. When mouse brain emulsions infected with neurotropic virus were centrifuged at high speed, the resulting sediment containing most of the infectivity of the original material demonstrated the interfering activity, while the supernatant did not. These findings support that the virus particle is the interfering agent.
7. Neurotropic virus inactivated by ultraviolet irradiation interfered with pantropic virus.
On the bases of these findings, the behavior of neurotropic virus in the extraneural organs was discussed and the possibility was suggested that there might be an infection process in which the virus can not complete the multiplication process to produce mature virus, but inhibit the multiplication of superinfected pantropic virus.

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