In the serological approach of typing various myxoviruses, fairly a variety of serum specimens, i.e. obtained at different stage of immunization from different animals, have been employed as the antibody without so serious precautions. However, in discussing the common antigenicity shared with various myxoviruses or in discussing the avidity of different antibodies to see the effect in experimental infections or in discussing the differea t susceptibility of antibody to the action of KIO₄, as was the case at the outbreak of asian flu, it was thought to be an urgent problem to get information on the nature of antibodies obtained at different stage of immunization.
Thus the study was conducted using allantoic fluid culture of PR 8 virus as an antigen. The antigen was injected one to chicken intravenously, the other to guinea pig both intracardially and intranasally. Antibody response was tested for every 9 weeks. Only hemagglutination inhibition test was used for the antibody assay, but in the elaborated manner where the titer against wide range of hemagglutinin was examined to find out the serum of different avidity.
The main result obtained in this study will be summarized as follows:
1) In chicken, highest antibody titer was obtained at first week after intravenous injection. The antibody here obtained was of high avidity and resistant to the action of KIO₄. Decrease in antibody titer as well as in avidity was found corresponding to the time elapse after the single antigen stimulus given intravenously.
2) In guinea pigs, even intracardial injection failed to produce antibody at early stage. 3 weeks were necessary to obtain the heighest titer, First week antibody obtained after intranasal injection was particular in its high sensitivity to the action of KTO₄ and in its specific activity against the PR 8 virus, in the background where the other serum specimens so far tested reacted not only to the PR 8 strain but also to WS strain of influenza A virus. The first week antibody here obtained was also of low avidity.
Reinfection of guinea pig 8 weeks after the first infection resulted in the early antibody response. Moreover, antibody thus obtained was of high avidity.

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Phage ε³⁴ is a temperate phage, capable of converting group E₂ Salmonella (O antigens 3, 15) to group E₃ Salmonella (O antigens (3), (15), 34) by lysogenization. The formation of antigen 34 is accompanied by partial loss of antigens 3 and 15, suggesting a close relationship between antigens 34 and 3 or 15 (Nakagawa, 1957; 1959).
Since Salmonella anatum (group E₁, O antigens 3, 10) does not adsorb phage ε³⁴, its lysogenization with ε³⁴ was carried out under particular conditions suggested by the experiments by Uetake, Luria and Burrous (1958). The resulting ε³⁴-lysogenic cells (=A(ε³⁴)) are same as S. anatum in their O antigens and in their sensitivities to phages ε¹⁵ and ε³⁴, excepting their resistance to phage C₃₄₁. When they are infected with ε¹⁵, both antigens 15 and 34 appear within several minutes in infected cells.
Most remarkable are the findings that A (ε³⁴) cells do not form antigen 34, irrespective of the presence of prophage ε³⁴, which always leads to the formation of antigen 34 when it infects 3, 15 cells of group E₂, and that antigen 34 appears when A (ε³⁴) cells form antigen 15 by infection with phage ε¹⁵ These observations, together with Nakagawa's finding mentioned above, indicate that the formation of antigen 15, at least partially, is a necessary prerequisite for the formation of antigen 34. Antigen 3 does not seem to be necessary for the formation of antigen 34, since antigen 3 of S. anatum is identical with that of group E₂ cells or of A (ε¹⁵) cells but there is neither formation of antigen 34 nor loss of antigen 3 in A (ε³⁴).
The resistance of A (ε³⁴) to phage C₃₄₁ is considered to be analogous to the resistance of lysogenic strains to unrelated phages, which has been observed in phages P1, P2 and λ (Bertani, 1953; Benzer, 1955).

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(1) By treating influenza A virus suspended in 0.1M phosphate buffer (pH 7) with 5×10^-4M p-chloromercuribenzoate (PCMB) for 24 hours at 37°C, the infectivity which was measured by the membrane piece technique could be destroyed without affecting hemagglutinating capacity.
(2) When an equal volume of an aqueous solution (pH 7) of the reactivating agent, cysteine or sodium thioglycolate, within the initial 30 minutes of the inactivation period, was added to the PCMB-treated virus, the reactivation was obtained. When cysteine or sodium thioglycolate solution was added after 60 minutes of the inactivation period, essentially no reactivation was obtained.
From these results, it seemed that SH group of virus protein was necessary to manifest the infectivity, and it was concluded that the infectivity and the hemagglutinating capacity were the independent attributes respectively.

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It was found that chloramphenicol, when added before T2 infection, blocked the formation of protein and DNA, but not the RNA synthesis after infection. The RNA synthesis after infection seems to be slightly stimulated by the addition of chloramphenicol. This RNA, which is formed without a priori formation of protein after T2 infection, is also characteristic of T2 infection in its mononucleotide composition. These results show that T2 infection induces a synthesis of new RNA, or at least a specific change in RNA metabolism at first without the prior synthesis of new protein.

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In an attempt to analyze the relationship between viral multiplication and the host cell metabolism, the effects of two metabolic inhibitors, that is, 5, 6-Dichlcro-1-β-D ribofuranosyl benzimidazole (DRB) and proflavine, upon one step growth cycle of ectromelia virus in L cell tissue cultures were studied. As the biological tests, infectivity (PFU), complement fixing antigen and the percentage of “B” type inclusions which proved to be the site of DNA and viral antigen were examined.
Morphological studies were also carried out on the inclusions. The dosage of 20γ/ml of DRB which is not toxic to cell reproduction for a certain period, could still suppress the formation of any of the three componeots mentioned above when administered 24 hours prior to virus infection. Inspite of the remarkable decrease in the number of inclusions, each inclusion formed developed, showing regularly granular and network structure just as the inclusions in untreated groups. However, DRB turned out almost ineffective when it was administered after the stage of virus synthesis. These results suggest that some disturbance of RNA metabolism in the host cell caused by DRB prior to the virus infection, might inhibit bit the synthesis of virus DNA and viral protein.
The dosage of 1mg or 2mg of RNase per ml. which does not affectt much the cell reproduction, was found to have a very similar effect on the viral multiplication to that of 20γ/ml of DRB.
The attitudes of these two substances concerning RNA metabolism towards the viral growth indicate that there might be an intimate relationship between the multiplication of DNA type virus and RNA metabolism in the host cells.
In the experiments with 0.5γ/ml of proflavine which does not affect the cell growth, only infectivity of virus was suppressed by both pre-and post treatment of virus inoculation. However, both complement fixing antigen titer and inclusion cell percentage were almost as high as those in the control. It may be of interest to note that the morphological development of inclusions formed was somewhat inhibited.
When more than 2.5γ/ml of proflavine which is destructive to the cell growth, was given at any stage after virus inoculation, infectivity was remarkably suppressed and most of the “B” type inclusions formed under this condition were small and compact, showing poor development of the inclusion bodies, in spite of the considerably high percentage of inclusion formation. However, all proflavine treated “B” type inclusions gave the same degree of Feulgen reaction and positive fluorescein antibody reaction as those of the control, showing the synthesis of these virus components. The “A” type inclusion (Marchal body) could hardly been observed by 16 hours.
These results seem to support the idea of De Mars that proflavine may interfare either with a hypothetical assembly mechanism or with the synthesis of some as yet unrecognized virus constituents.

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