(1) From 1954 to 1956 the authors isolated 51 strains of cytopathogenic agents mostly from feces of patients with paralytic poliomyelitis using human embryonic skin-muscle tissue culture. As a result of typing we found 31 strains of type I (70%), 7 strains of type II (15%), 7 strains of type III (15%) and 6 untypable agents. 70 to 80 per cents of causative agents of paralytic poliomyelitis belonged to type I in Japan in the above period.
(2) Neutralization test using 100 TCD 50 of three types of poliovirus vs. undiluted and diluted (1:10) sera from healthy individuals was done on 209 specimens from Tokyo. The percentage positive was lowest at 6-11 months after birth, increasing rapidly, and the adult level (80-100%) was reached about 4 to 6 years of age. This pattern of the age distribution of antibody is almost equal to those obtained in Cairo, Egypt, and French Morocco. In the case of diluted sera the final level attained was 60-80 per cent, so that about 20 per cent of immune adults were considered having antibody titer less than 1:10.
(3) 46 per cent of paralytic polio occurred in the first year of life in Tokyo 1955, and steep increase of neutralizing antibody was seen among examinees belonging to the same age group. It means that paralytic sequelae are very likely to occur in the primary infection without protection of antibody.
(4) Analysis of antibody for one or more types of poliovirus in various age groups shows that infection by any one type interfere with heterotypic infections, in other words there exists some degree of cross immunity between heterotypes.
(5) Antibody titers of the pooled sera which were composed of an equal quantity of sera from individuals belonging to the same age group were minimum (antibody titer against type II and III were zero, but that of type I was 1:3 to 1:8) under one year of age, rose up to maximum (1:64 to 1:128) at 4 to 6 years of age, then declined a little and kept at a level (1:16 to 1:32) thereafter in Tokyo.

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By inoculating the HVJ (Z) virus together with the influenza (A) PR8 virus into the chorioallantoic cavity (CAC) of a developing embryonated egg, they were found to multiply in mixed existence. To obtain the ratio of one strain to the other thus multiplied in mixed existence in the chorioallantoic fluid (CAF), two different methods were employed. One was to utilize the heat fragility of the Z virus and determine the chicken cell agglutinating titers (CCA titers) both before and after the CAF was heated in hot water of 50°C for ten minutes, while the other was to add antiserum to the CAF and thus determine the inhibition titers. When the inhibition titers were determined, however, the inhibitory effect shown by some of them was such as could never be detected in the control experiment in which only the two strains were mixed artificially. Furthermore, the ratios of the two strains in simultaneous growth, when obtained by each of these two different methods, were found to disagree.
The cause for such disagreement and unusual inhibition titers may be considered to be either (1) the changed heat fragility or (2) changed antigenicity of the strains. These variable strains with changed heat fragility or changed antigenicity, however, were found to revert to the pure Z virus or PR8 virus in its original form in the course of serial passages. Consequently, it was impossible to preserve their peculiar characters generation after generation by means of limiting dilution passage.

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As described in the first report, both the HVJ virus and the influenza (A) PR8 virus were made to multiply simultaneously by mixed inoculation of them into the CAC of a developing embryonated egg, and examined by two different methods, one taking advantage of the heat fragility of the Z virus and the other adopting as a means the determination of the hemagglutinating inhibition titers (HI titers). The result was that the ratios of the two strains in simultaneous growth, when obtained by each of these two different methods, were found to disagree and that there were some variable clones with such inhibitory effect as could never be found in the control experiment in which only the two strains were mixed artificially. This was probably because both the thermofragility and antigenicity was subjected to change, and the variable clones thus produced might possibly be considered to be some recombinants of the two strains. But since most of these variable clones were found to revert to the pure Z virus or PR8 virus in its original form in the course of limiting dilution passages, it is impossible to say definitely that their peculiar characters are entirely attributable to their gene.
Both the change of thermo-fragility and that of antigenicity can be considered to be phenotypic. As a different type of progeny is sometimes produced even by variable clones of the same phenotype, the cause for it may be that virus particles of different types mixed in the clones are put together and thus manifest a single type. In an attempt to inquire further into the characters of these clones, the author analysed the characters of the progeny produced by them in the course of serial passages in order to find the constituents of the parent strain, and parallel with it, made a statistical observation of the characters of those descendants emerging from each variable strain.

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As previously reported, the HVJ virus and the influenza (A) PR8 virus were made to multiply simultaneously in the CAC of a developing embryonated egg, and upon examination of the multiplied strains by the CCA test utilizing the heat fragility of the Z virus and by the inhibition test with the aid of antiserum, the heat fragility of the CCA activity and the antigenicity of the strains were detected to undergo some change. This is phenotypic mixing, but as most of the clones showing such phenotypic mixing were found to revert to the pure Z virus or PR8 virus in its original form in the course of serial passages, it was impossible to preserve their characters generation after generation by means of limiting dilution passage. This might be considered to be probably due to a very slight affinity existing between the Z virus and the PR8 virus. Instead of the PR8 virus, therefore, the NWS virus regarded to be weaker in its resistance to various sorts of experimental processes and to have characters highly similar to those of the Z virus was taken up, and by mixed inoculation of these two strains so as to make them multiply simultaneously, isolation of viruses in a recombinant form was attempted.
The result proved the same then as when the PR8 virus was employed. Clones clearly showing the phenotypic mixing of the NWS virus and the Z virus could be produced, but as these clones of this type were found to revert to the pure Z virus or NWS virus by means of limiting dilution passages, it was impossible to preserve their characters generation after generation. The author is uncertain as to whether such viruses showing phenotypic mixing carry abortive infection or have a strong inclination to revert to the pure, original form, and it is hardly possible to say definitely that this phenomenon of phenotypic mixing was caused by some recombinant form of virus clearly attributable to the gene of these strains.

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The unusual characters of the HVJ (Z) virus considered to belong to the MNI group were described in the previous reports. In the present experiment, an inquiry was made into the possible interrelations between its blood cell agglutinating activity, eluting activity and hemolytic activity.
When the purified and concentrated virus preparation was resuspended in various kinds of media regulated to pH 7.2 and the virus suspensions thus obtained were kept in hot water of 45°C, it was found that each solution of 4%-2% casaminoacid, 1%-0.5% broth, 2%-1% polypepton, and 0.05 mol of Lysine-HCI could strongly protect the CCA activity, while glucose, sucrose and dextran solutions could not. Urea had an inactivating effect on the CCA activity. Lysine was found capable of being adsorbed onto the Z virus. It is possible that the fact might have something to do with the heat stability of the CCA activity.
Heating at 45°C reduced the infectivity, but this reduction of infectivity was comparatively small in the case of a medium capable of protecting the CCA activity. Furthermore, when the virus was kept either in a casamino-acid or a polypepton solution at 45°C for 16 hours, the CCA activity remained normal but the infectivity disappeared almost completely. Yet, as its capacity of being eluted from red blood cells was retained, a sample of low I/A ratio could be produced, but the indicator virus was hard to obtain.
Regardless of the kinds of media in which the virus is suspended, its hemolytic activity disappears by about 2 hours heating process. As stated above, the red blood cell agglutinating activity and eluting activity of the virus can be protected by certain kinds of media in which it is suspended, but with regard to its hemolytic activity, the case is different. It is evident, therefore, that the hemolytic activity is in no way related to the CCA activity or eluting activity.

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In the first report, the studies on purification and concentration of GDVII virus by means of hemagglutination procedure were described. However, that the concentration of the virus by this procedure did not produce satisfactory yields of virus because of hemolysis appearing in the process of concentration of the virus by elution from erythrocytes. The purpose of the present paper is to report the research on more effective method by means of adsorption to and elution from the red cells stroma which are capable of adsorption of virus.
In the first step, the virus was adsorbed to the human O Group erythrocytes, and was eluted from erythrocytes by saline solution in the volume which did not occur hemolysis, and finally the virus eluted into the saline solution was adsorbed to and eluted from the twenty per cent stroma suspension. The results obtained indicated that the concentration procedure of virus by stroma was more effective than that of erythrocytes for the removal of total nitrogen in the final product, i.e. the former was able to concentrate the virus in more approximately 1×10⁶ hemagglutinating units per mg N, and 1000-fold LD₅₀ per mg N than that of the original materials.

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In the studies on the interference phenomenon in infectious ectromelia of mice, the following results were obtained. UV-irradiated virus was found to be more potent in its interfering effect than formalin-inactivated virus and the virus inactivated by heating at 56°C for 5 minutes was shown to have lost its interfering effect.
The agent active in the interference was shown to be in virus particle as already observed in the other viruses and the Seitz-filtrate which contained no virus particle did not have interfering effect.
When inactivated virus was injected within 3 hours after challenging with living virus, interference took place. In the experiments with various inoculation routes and varying concentrations of inactivated virus, when living virus was inoculated immediately after the injection of inactivated virus, the intravenous injection of both viruses gave more effective interference than the intraperitoneal injection, and the intravenous injection of even low concentrations of inactivated virus gave a good interference.
Mice were cross-infected with Rift Valley fever virus and ectromelia virus and the interference between inactivated virus and living virus was studied. The interference was found only in the combination of homologous virus preparations. The serum antibody appeared in the mice only after 20 days following the injection of inactivated virus. When living virus was inoculated at varied intervals after the injection of UV-irradiated virus, two groups of survived mice were differentiated. One group which received inoculation shortly after the injection of inactivated virus was thought to have been saved by interference and the other group which was challenged more than 20 days after the pretreatment was considered to have been protected by antibody. Through this experiment, interference phenomenon was assumed to be based on a mechanism different from immunity due to antibody.

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The purpose of this paper is to report a part of an exploratory experiment to ascertain the presence or absence of assimilar modifying effect of bacteria or bacterial substances on the course of neurotropic virus infection as that of pneumotropic virus in the natural state.
The GD VII strain of mouse encephalomyelitis virus was used as a virus, and Staphylococci, Sarcina lutea, Streptococci, Diplococcus pneumoniae, H. pertussis, Proteus OX19, Brucella abortus Bang, E. Coli, S. Typhi, Shigella dysenteriae, S. cholerae suis, V. cholerae, and Myc. smegmatis were used as bacteria. Groups of 3-4 weeks old mice were inoculated intranasally with cultured bacteria mentioned above, in approximatly 0.03ml two days, and one hour before and two days after, respectively, inoculation with dilutions of GD VII virus by intranasal route under ether anesthesia. The effect of bacteria on the course of virus infection was estimated by the degree of reduction or increase in the LD₅₀ of the experimental group from the level found in a comparable group of mice which had received with sterile broth.
The results obtained indicate that Sarcina lutea and S. cholerae suis have the stimulating effect of virus infection and E. coli, H. pertussis, and V. cholerae have the inhibitory effect; E. coli, in particular. The nature of the mechanism of these effects will be clarified in the following extensive studies.

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We studied the hemagglutinating activity, infectivity and immunizing ability of influenza virus FM1 (egg-adapted strain and mouse-adapted strain). The mouse-adapted strain FM1 had infectivity, immunizing ability and toxicity against chick embryo proportion to its hemagglutinating activity, but not against mice.
After we repeated the adsorption of virus in virus-suspension with chick red cells, we inoculated the 10^-1-10^-5 dilution of that non-hemagglutinating supernatant into the chorioallantoic cavity of eggs, and, after 48 hours' incubation, we recognized the virus-production by their hemagglutination. So that there may be the non-hemagglutinating but infective influenza virus.

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