It is well known that interest informations on the antigenic structure of viruses have been obtained with complement fixation. The serological properties of Newcastle disease virus, however, have been mainly studied by neutralization and hemagglutination and available references on its complement fixing properties are limited. For complement fixation many kinds of animals may be able to donate antibodies, but the natural host should be considered to be standard as source of antibodies. As chicken serum, which is of the most susceptible natural host for the virus, fails to fix complement when inactivated by heating at 56°C for 30 minutes, indirect complement fixation has been recommended as an available technique. Recently the possibility of direct complement fixation with chicken serum was suggested by Nitzschke (1956) and Brumfield (1957). The author tried to establish the direct complement fixation in the Newcastle disease virus-chicken serum system and to examine complement fixing properties of the virus and antigenic difference among several strains with the technique.
The Sato, Miyadera and W strains of Newcastle disease virus, different in origin and pathogenicity, were chiefly employed. The Sato strain was isolated from an affected bird in the outbreak of the acute type disease in 1930 and the Miyadera strain, of the mild type disease in 1951. The W strain, originating from American living vaccine, is an avirulent one in chickens.
Results are summarized as follows.
1. Direct complement fixation was successfully performed in the Newcastle disease virus-chicken serum system, using sera in the active phase and the incubation at 37°C for 60 minutes in a water bath.
2. Complement was specifically fixed with infected embryonic tissues under the presence of immune serum. The highest fixation was shown in the test with chorioallantoic membranes and no fixation was detected in the test with allantoic fluid.
3. Two kinds of antigens, soluble and viral, existed in infected embryonic tissues. The former was prepared by the complete elimination of hemagglutinin from the suspension of chorioallantoic membranes, using centrifugation and absorption with chicken red cells, and the latter, by centrifugal concentration of hemagglutinin from allantoic fluid.
Although hemagglutinability was approximately the same in titer, the amount of soluble antigen in chorioallantoic membranes seemed to be different among the strains.
4. Antibodies against soluble and viral antigens were produced in chickens hyperimmunized with inactivated virus or infected with active virus. Antibody against viral antigen was usually from 2 to 8 times higher than that against soluble antigen in titer.
5. Partial antigenic differences among the strains were revealed by cross hemagglutination inhibition with sera absorbed with heterologous antigens, particularly the Sato strain appeared to be different from the others. Cross complement fixation failed to detect the partial differences among them, even when viral antigen was used. The Miyadera and W antigens, both soluble and viral, showed stronger reaction with the Sato immune serum than that with the homologous. The Sato strain seemed to be more effective to produce antibodies in chickens.

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It has been known that equine infectious anemia caused by a virus. The experimental study of this disease has been severely handicapped by the lack of suitable experimental animals susceptible to the virus. It would facilitate the study of the disease enormously, if the virus could be cultivated in vitro. This has not been possible so far.
The experimental study of the virus cultivation through tissue culture method was carried out by the author since 1955.
The primary results are described in the present paper. Eight experiments of virus passage were performed respectively using various horse tissue with three culture methods. The plasma clotting roller tube culture (RT), the trypsin-dispersed monolayer cell culture (ML) and Maitlandtype tissue culture (MT) were employed.
The mixture of 30% horse serum, 10% CEE and 60% Hanks' BSS or 30% bovine serum and 70% synthetic medium No. 199 were used mainly as nutrient media. Infected horse sera (Goshun and Wyoming strain) collected at the acute phase were used as the original virus material. The material was inoculated into the culture in which excellent outgrowth was shown (RT and ML), or into the culture immediately after the cultivation (MT).
The serial cultivation and horse inoculation test were performed using the cultured materials. Cytopathic changes of the inoculated culture were observed microscopically under low magnification (RT and ML). The results are as follows.
1) Outgrowth of 16 kinds of tissues in RT culture exhibits different growth rate and cell type. Spleen, lymph node, Testis, and others showed active growth of fibroblast-like cells, while epithelial cell growth was manifested in kidney, lung and liver.
2) In four serialcultivations (RT, Exp. 1-4) using several series of spleen, lymph node, kidney, lung and adrenal gland, 4 or 5 and 7 day intervals of virus passages were carried out until 3-8 generations. In a series of kidney ML culture (Exp. 5), the virus passage was also performed until 4th generation adopting 4-5 days' interval. No cytopathic changes were observed through the experiments. The results of horse inoculation tests of the cultured materials produced positive infection only first-third generation. From the results, it may be presumed that total dilution of test material induced positive infection did not over the infective titer of original inoculum. Therefore, it was considered that the virus growth was negative in those culture.
3) In the serial passage of Exp. 6 (MT) including the series of spleen, lymph node and adrenal gland, 10 days' interval was adopted, and 2nd generation of the cultured material resulted positive infection by the horse test, but advanced passage materials showed negative. However, in two series of the virus passage using liver MT culture which were adopted 14 day (Exp. 7) and 7 days' interval (Exp. 8), the 4th (Exp. 7) and 8th (Exp. 8) generation of cultured materials caused positive infection respectively. According to the results, in the liver MT culture, it was suggested that the growth of the virus was rather positively in the relation of original virus infective titer.
However, the cultured material in advanced passage resulted negative, and furthermore, 8th generation material which induced a positive infection in primary test resulted negative by the retest at 6 months later.
The results were considered that the virus in the cultured material was inactivated during the preservation under unsuitable condition especially in a high level of pH.
4) Three samples of virus sera (Wyo strain) including the one used in the series of experiments (Exp. 4, 6, 7 and 8) resulted positive infection respectively at 10^-5 dilution, but was negative at 10^-7 by the horse inoculation tests.

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In the previous paper, the author reported on unfavorable results of serial cultivations of equine infectious anemia virus using the cultures of various horse tissues in several methods. In this experiment, horse bone marrow culture was examined for the virus propagation. Bone marrow were collected mainly from femoral bone of healthy colts, and trypsinized. The resulting cells were suspended into the nutrient medium in concentration approximately 10⁷ per ml. Two ml of the suspension was placed into each modified carrel flasks and cultivated stationally at 37°C. Medium exchanges of the whole volume were carried out at every 1-3 days. For some experiments, the punctured bone marrow fluids were used, also, as a source of the cell culture. The mixture of 40% bovine serum and 60% synthetic medium No. 199 were employed as nutrient fluids in the majority of experiments, and the mixture of 30% horse serum, 10% CEE and 60% Hanks' solution were used in some cases. The infected horse sera (Wyo. strain) collected on the acute phase were used as original virus material, and healthy control sera were obtained from the same horses at immediatly before the virus inoculation. The both sera were inoculated 0.2ml into each cultures at 1 day after the cultivation, and serial passages were performed in parallel with each other. The cultures were observed microscopically under low magnification.
Results obtained are as follows.
1) Two kinds of cells in the bone marrow culture were morphologically observed. The one is round cells which was not ascertained its multiplication in vitro and cultivated on the glass surface for 2-4 weeks or more, while, the other is fibroblast-like cells which appeared 2-4 days after the cultivation and showed active cell growth. Cultures of punctured bone marrow fluids resulted poorly culture of round cells, but fibroblast-like cells were easily cultivated showing the active growth.
2) The cultures inoculated with virus sera manifested the cytopathic change after 1-2 weeks of the virus inoculation. The C. P. change was observed only in the round cells and no change was recognised in fibroblast-like cells.
3) The fluids which were harvested from cultures exhibited C. P. change reproduced the similar change in the following renewed cultures by transfer inoculation, and 8 passages through 81 days were performed by checking sign of the C. P. change.
4) Horse inoculation tests were performed using the cultured materials of 1st, 2nd, 3rd, 5th, 6th and 8th generation of virus passage series and of 1st, 5th, 6th and 8th generation of control passage series. Test horses inoculated with each materials of virus series infected positively in dosis of 1.0-0.001ml, but whole the ones inoculated with control series materials manifested no signs of infection injecting 1ml dosis of test material.
The dilution of the inoculated material of the last generation which indicated positive infection was calculated 10^-12 of original inoculum, even with the exception of 40 times exchange of medium in vessel, and was contrasted with the virus titer of the original inoculum, which was infective at 10^-5 dilution and not at 10^-7. From the above results it would be confirmed that the virus propagate in horse bone marrow cell culture.
5) The serial passage of the virus using the cultures of fibroblast-like cells from bone marrow fluids was advanced until 3rd generation adopting 14-16 days' interval, but the cultures showed no C. P. changes, and the horse inoculation test using the cultured material resulted positive in only the first generation and negative in further transfer.
As the results mentioned above, it may be considered that equine infectious anemia virus is able to propagate in the round cells with the indication of cytopathic changes, while, it seems to be negative in fibroblast-like cells which showed active growth in the culture.

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