In the previous paper the author reported that the propagation of equine infectious anemia virus was noticed in trypsinized bone marrow cell culture showing the cytopathic changes of the round cells. The present paper describes that the virus propagation was also comfirmed in the cultures of peripheral blood leucocytes of horse. The leucocyte culture was performed by following method. The heparinized blood was collected from jugular vein of healthy young horses, and allowed to stand at room temperature for 15-20 minutes, then the plasma was removed. Leucocytes were collected from the plasma by low speed centrifugation, and washed once in cold Hanks' solution. Then, the leucocyte sediment was resuspended into the nutrient medium in concentration of approximately 10⁷per ml, and cultivated stationally at 37°C. Modified carrel flask was used containing each 2ml of the cell suspension. Mixture No. 199 with 40% bovine serum was used as nutrient medium.
Infected horse sera (Wyo. strain) collected during the acute phase were used as original virus materials, and healthy control sera were obtained from the same horses at immediately before the virus inoculation and the both serum materials were used in parallel with each other.
Virus inoculation, observation of the culture and serial cultivation were performed by mean of bone marrow culture method described in the previous report.
Results obtained are as follows.
1) Horse leucocytes were cultivated on glass wall during 2-3 weeks, but the cell multiplication was not recognized.
2) On 7-9 days after the virus inoculation, the cultured cells manifested the similar cytopathic changes to the ones which ascertained on the round cells of horse bone marrow culture.
3) The fluids harvested from virus inoculated cultures which appeared the C. P. changes reproduced the similar changes in the following cultures by transfer inoculation. Consequently, serial cultivation of the virus was carried out through 154 days in 17 generations by checking sign of the C. P. changes. In despite of the lack of C. P. changes in control culture inoculated with the healthy horse serum, the culture fluids were transfered successively in parallel with that of virus passage series.
4) Horse inoculation tests were carried out using the serially cultivated materials, and results obtained are as follows. Testing each 1ml of the materials of 7th and 14th generation and 1ml of 10^-1 diluted material of 17th generation in the virus passage series the infectivity against horse were demonstrated as positive, but, 1ml of the material of 14th and 17th generation in the control passage series did not induced any sign of disorder in inoculated horses. The dilution of the last inoculated material in the virus series was calculated 10^-23 of original virus material by serial passages except the medium exchange in total 70 times performed during the successive serial cultivations.
As the results described above, it may be concluded that equine infectious anemia virus propagated successively in horse leucocyte culture and appearance of cytopathic changes would be accompanied with the virus propagation.

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Attenuation of the Okuda and Kinugasa strains of influenza A2 virus, isolated in our laboratory in 1957, was attempted by egg passage, using chiefly 10 or 11 day eggs. Mainly 10^-3 dilution of infected chorio-allantoic fluid was used as inoculum for serial passage. Infected chorio-allantoic fluids of various generations of egg passage were inoculated by inhalation or instillation method to human volunteers. For inhalation, an outlet of a nebulizer with an air compresser was kept 1-2cm apart from the nostrils of volunteers, and fine mist was inhaled by deep or normal breathing for 10 or 60sec. Instillation method was far less efficient than inhalation method, and only inhalation method was used in the following experiments.
The Okuda strain of the 3rd and the Kinugasa strain of 7th egg passage provoked typical signs of influenza. The materials of about 100th passage of these strains proved to have little pathogenicity, while antigenicities of them were well preserved (Table 1, 2).
Egg infectious titer of materials and inhalation time were important factors. When the titer was low, hemagglutination inhibiting (HI) antibody was poorly provoked in the subjects. When the titer was high and the material was inhaled for 60sec., some persons showed transient fever without local catarrhalic sign. When the inoculum was not so high, general toxic reactions were negligible (Table 3). Not only HI antibody, but other antibodies increased (Table 4). The persons with clinical signs such as fever, headache or lassitude, showed far poorer antibody response, than those without clinical sign.
Reisolation of virus from inoculated persons was unsuccessfuland infection to others from them was not observed. The S-antibody rise and the small quantities of virus needed for antibody response seem to show active propagation of influenza virus in human volunteers. No significant difference was observed between these two strains.
The strains and the inoculation method are hopeful to be used as live influenza vaccination.

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In the first paper of this series work, the inhibitory effect of the antibody produced at the time of primary infection on the virus growth at the late stage of infection was suggested. It was particularly the case at the time of intraperitoneal injection of a large amount of virus. In this study, detailed examinations were made on the growth characteristics of the same PR8 virus in lung after intranasal inhalation and again the effect of antibody on the virus growth was tested.
Seventh report following this paper will describe the production site of antibody against the PR8 virus and 8th paper may concern on the histopathological examination of the lung, particularly when the mice was infected intraperitoneally. When the descriptions in these series papers, from 6th to 8th, are taken together, significance or limitations of the immune mechanism to the development of infection will be revealed.
Main results obtained in this work will be summarized as follows:
1) Effect of the small amount of inoculum given by inhalation on the virus growth in lung and on the fate of mice was examined. As a result, 10^2.7 EID₅₀ particles were found to be necessary as the 50% lethal dosis. 10^1.2 EID₅₀ particles were necessary in inducing infections in 50% of the mice. Whenever the virus growth in lung was detectable, consolidation of the lung always followed.
2) At the time of intranasal infection, the initial antibody rise was fairly postponed when compared to that after intraperitoneal infection.
3) The decrease of virus titer in the lung exactly followed to the shift of serum antibody titer, when the small inoculum size was given by inhalation. This kind of keen correlation has been already secured at the time of intraperitoneal infection.
4) Virus growth at the time of intraperitoneal infection was also involved in the study to characterize the virus growth at the time of intranasal infections. Even when the large amount of virus was inoculated intraperitoneally, the inoculum actually participated in the multiplication in lung was estimated to be quite small and the initial rise in virus growth was postponed 6 hours when compared to the growth curve obtained at the time of inhalation.
5) Antibody response of mice receiving intraperitoneal injection of formaline-inactivated virus was also studied. On the basis of this observation, an experiment was conducted to see the participation of immune mechanism on virus growth. To do this, inactivated virus was injected intraperitoneally on the first day, then the sublethal amount of alive virus was inhaled on the second day. Growth of virus and consolidation of lung in these animals were almost similar to those obtained when the alive virus was given intraperitoneally. In other words, immunization effect of the formaline-killed virus given one day ahead was clearly demonstrated against the mice which received intranasal infection of small amount of virus.
6) To confirm the participation of viral antibody to the virus growth, radiation effect was studied with mice infected intranasally with small inoculum. With irradiated animals, virus growth continued longer than the non-irradiated.
Reviewing our experimental results as a whole, it is broadly true that the antibody response of the mice at the time of primary infection influences the virus growth in mouse lung, at late stage.

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Antibody level in the serum of mice infected with influenza virus via three different routes has been already described. In discussing the participation of immune mechanism on the growth of virus as well as on the fate of experimental animals, rather the series of events accompanied to the establishing process of immune status was thought to be a matter of interest. Having this kind of consideration in mind, antibody titers in various organs of infected mice have been pursued by means of hemagglutination inhibition test.
Prior to the experiment, the procedure to remove all of the non-specific hemagglutinin inhibitors from various organ extracts was examined. This was easily done by treating the specimen with cholera filtrate, even when the heated Lee virus was used as an indicator. Then the antibody titer against the PR8 virus, the strain which had been known to be most insensitive to various α-inhibitors has been examined.
Main results obtained along the study will be summarized as follows:
1) The amount of α-inhibitor both in lung and bronchial washings after intranasal or intraperitoneal infection has been pursued. Inhibitor titer curve obtained against the day was just a mirrorimage to that of virus growth.
2) After the inhalation of sublethal amount of PR8 virus or after the intraperitoneal injection of large dosis of alive or formaline-inactivated PR8 virus, the antibody titers of serum, bronchial washing, lung, liver and spleen were examined at appropriate intervals. To visualize the production curve of antibody in any organs concerned, a value “antibody index” was calculated. When antibody titer in some organs at some stage was taken as a numerator, and antibody titer in the sera at the same stage was taken as a denominator, this value was obtained.
Only in spleen, the value of antibody index was higher than 1 at an early stage of infection or immunization. This kind of early antibody rise in spleen was never observed at the time of passive immunization when rabbit serum prepared against PR8 virsu was injected intravenously.
3) Consolidation parts of the lung found at late stage of infection was also believed to be responsible for the antibody production. The value of antibody index obtained with consolidation parts was apparently higher than that of healthy parts of the same lung, and was almost 2.
4) However the antibody found in bronchial washing was thought to be of different nature when compared to that in lung. With the former, the increase in titer was demonstrated by nonspecific stimuli, whereas the fact did not hold true with the latter.

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1. A lysate of a high T3 titer was obtained by the following procedure. Escherichia coli B of logarithmic phase was centrifuged down and resuspended in a fresh medium at the concentration of ca. 5×10⁹/ml and infected with T3 (multiplicity of infection =1/20) and shaken at 37°C. The phage titer of the resulting lysate reached usually 3-5×10¹¹/ml.
2. T3 can be concentrated from the lysate by ammonium sulfate precipitation or evaporation under reduced pressure.
3. Highly purified T3 was recovered from DEAE-cellulose chromatography. A DEAE-cellulose column, washed thoroughly with a buffered saline (0.1M NaCl, 0.005M Tris pH 8), was charged with T3 suspended in the same solution. The column was washed with the same saline and T3 was then eluted with 0.2M NaCl, 0.005M Tris pH 8 in a good recovery.
From the experiments of the isotope labeling, the phosphorous content of T3 was calculated to be 6×10^-12γ P per plaque forming unit. The ultraviolet absorbancy of T3 was also determined. The optical density at 260mμ was 0.18 with the suspension of 10¹¹/ml.
4. The stability of the phage was investigated. T3 was less stable at low salt concentration and acidic pH (under pH 6).

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1. It was very difficult to extract the deoxyribonucleic acid from purified coliphage T3 by the usual phenol method. But after acid treatment, T3 DNA became easily extractable by shaking with phenol. A suspension of T3 was first acidified to pH 4.7 by the addition of 0.1M acetate buffer and stood for 10 minutes and then neutralized to pH 8 with 1M Tris pH 9.3. An equal volume of freshly distilled phenol was added and the suspension was shaken for 15 minutes and centrifuged. From the water layer of the centrifugate, DNA was recovered by the usual procedure in a good yield.
2. This preparation was free from any protein and its value of ε(P) was 6400 at 260mμ.
3. The base composition of this preparation was determined by quantitative paper chromatography. Its base ratio was G:A:C:T=25.8:24.0:24.0:26.2

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1) Crude extracts were prepared from T2-infected E. coli which incorporated S³⁵O₄^--for a few minutes during the initial reaction period. The extracts were fractionated by starch zone electrophoresis, with ammonium sulphate, and by ultracentrifugation. Distributions of protein-S³⁵ (“early protein”) among several fractions were studied and compared with that of protein-S³⁵ (“coli type protein”) from crude extract of uninfected bacteria which were similarly treated. No difference of distributions has been observed with respect to the distribution of protein-S³⁵ between the early protein and coli type protein.
2) Heavily ultraviolet light-irradiated bacteria lost the ability to assimilate S³⁵ into proteins, while such bacteria infected with T2 phage assimilated S³⁵ actively because of reactivation. Analysis of the early protein-S³⁵ In T2-reactivated bacteria exhibited identical distribution of S³⁵ as compared to distribution of S³⁵ assimilated by uninfected bacteria.
3) In the identical condition of fractionation, striking abnormalities with respect to distribution of early RNA were observed as compared to that of coli RNA-distribution.

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