Japanese B encephalitis virus is one of the major members of the group B arbor (arthropod-borne) viruses, and it has a great significance in Japan. A number of studies on Japanese B encephalitis virus have been undertaken since long ago, but many physico-chemical problems still remain unsolved in comparison with some other animal viruses.
This paper reports the experimental results which tried to partially purify Japanese B encephalitis virus particle by means of three biochemical techniques.
Results obtained are as follows:
1) Japanese B encephalitis virus (G1 strain) used was grown in trypsinized hamster-kidney cell cultures. The culture medium consisted of 0.5% lactalbumin hydrolysate, 10% bovine serum, 100u/ml penicillin, and 100γ/ml streptomycin in Hanks' balanced salt solution. The crude virus concentration in culture fluid was about 10^5.0per 0.02ml in mouse-intracerebral LD₅₀, and it was obtained always four days after the inoculation.
2) Culture fluid harvested from the virus-infected cultures, showing characteristic cellular degeneration, was centrifuged at 3, 000r.p.m./min. for 10 minutes to remove crude cell debrises. The supernatant fluid was loaded onto a column containing ECTEOLA-Cellulose and eluted with 0.07M phosphate buffer solution (pH 7.2). Some supernatant fluid was treated with fluorocarbon. By either procedure 95-100% of the virus was practically recovered, and also more than 90% of the nitrogen contained in the original virus material, as measured by micro-Kjeldahl method, was removed. Appreciable partial purification in terms of protein removal by cholesterol column chromatography was also performed.
3) It was finally concluded from the above results that the procedures of ECTEOLA-Cellulose column chromatography or fluorocarbon deproteinization were the simple and convenient biochemical method which can be successfully applicable to the partial purification of Japanese B encephalitis virus particles.

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The incidence of enterovirus infections has been studied in a total of 58 apparently healthy infants and children under 3 years of age during one year from May 17, 1960 through May 18, 1961, in Takatsuki, Japan.
81 strains (4.7%) of enterovirus and 4 strains of adenovirus were isolated from all 1729 fecal specimens collected about every 10th day.
The monthly isolation rates showed a seasonal prevalence with its peak in August (16.6%), July (12.8%), September (9.9%) and October (7.5%), on the contrary the rates from November to May the next year were as low as 0 to 2 per cent.
The results of identification of the isolated viruses were as follows: Coxsackie B-5 strains were predominantly isolated, i.e., 30 of 53 strains of Coxsakie B viruses isolated were identified as Coxsakie B-5 virus, 13 and 10 viral strains belonged to ECHO and polio virus respectively and 5 strains remained untypable.
The evidence of infection with the enteroviruses were confirmed in 29 persons among all 58 examinees.
Of 29 who were infected with “enteroviruses”, 7 were proved to have been affected with two to five different types of the virus during one year of my study. Coxsackie B virus infections were verified in 24 among those 29 individuals, especially, Coxsackie B-5 infections were predominantly found in 15 persons.
There was no significant difference in the isolation rates by age, so far as my study was concerned.
The duration of virus excretion varied from 10 days to 2 months in polio and ECHO viruses, and from 10 days to 3 months in Coxsackie B-5 virus.
The neutralizing antibody test against Coxsackie B-5 virus was examined on two serum samples taken in May 1960 and January 1961. These 50 examinees gave negative neutralization test against Coxsackie B-5 in the prior sera, except for one who gave positive. 11 individuals of 50 examinees excreted Coxsackie B-5 virus and showed positive conversion of antibody to this virus in the second sera, and one individual showed a rising of the antibody titer but the fecal specimen was not obtained.
A clinical illness was not found in persons whom the enterovirus were recovered in this study. However, the epidemics of aseptic meningitis due to Coxsackie B-5 virus were reported elswhere in the Kansai area during the summer of 1960.
From the above mentioned results, it was confirmed that in Takatsuki not only poliovirus but also Coxsackie B viruses, especially Coxsackie B-5, were prevalent among infants and young children, particularly during the summer of 1960.
When considered the interference-phenomen between heterotypic enteroviruses, it is concluded that the most effective oral vaccination of polio should be performed during the season when the enterovirus infection is considered rare in a temperate zone like Japan.

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Phage ε¹⁵ propagated on cells of Salmonella anatum (=A) shows efficiency of plating (EOP) of about 10^-2 on cells of Salmonella butantan (=I-1), while ε¹⁵ propagated on cells of I-1 shows EOP of 10^-4 on cells A. The some is true in phages produced by respective lysogenic derivatives. One cycle growth of phage on one strain results in production of phages with lower EOP on the other. This reciprocal shifting of EOP has proved experimentally to be due to host-controlled variation (HCV) and not to host range mutation by the following experiments.
Phages can be adsorbed well even onto cells with lower EOP and phage genetic material is injected into the cells, resulting in de novo synthesis of somatic antigen 15 on infected cells. Some of cells may revert to sensitive cells even after being injected with genetic materials of restricted phages.
When cells I-1 were mixedly infected with ε¹⁵ [A] and ε¹⁵vir [A], number of mixed yielders was far more than that calculated under the assumption that each of ε¹⁵ [A] and ε¹⁵vir [A] contains a host range mutant capable of attacking cells of I-1 at a proportion of 10^-2 and 2×10^-2, respectively.
On the other hand, it has been shown that EOP is partly dependent on physiological states of cells and also that multiple infection may lead to phage multiplication even in cells which do not allow phage growth when singly infected.

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In the first paper of this serial works, the author reported that crude Japanese B encephalitis virus preparations were successfully purified by techniques of column chromatography with ECTEOLA-Cellulose ion-exchanger or by fluorocarbon deproteinization.
In attempt to develope these findings on Japanese B encephalitis, the present paper describes that some other arbor viruses and poliovirus were also partially purified by cellulose chromatography or fluorocarbon deproteinization.
Results obtained are as follows:
1) The viruses used were dengue fever type 1, Mochizuki strain; yellow fever, strain 17 D; and Western equine encephalitis, Rockefeller Institute stock strain. The viruses were also grown in trypsinized hamster-kidney cell cultures. The concentration of each crude virus in infected fluid was about 10^5.0 (mouse intracerebral LD₅₀ per 0.02ml) in the case of dengue fever, 10^4.0 in yellow fever, and 10^7.0 in Western equine encephalitis respectively.
Poliovirus, strain MEF-1, was used for comparison and grown in HeLa cell culture too. HeLa cells were grown in culture bottles with medium consisting of 2% yeast extract, 0.5% lactalbumin hydrolysate in Earle's balanced salt solution (YLA medium), and 20% bovine serum.
2) Culture fluid harvested from the infected cultures, showing characteristic cellular degenerization, was centrifuged at 3, 000r.p.m./min. for 10 minutes to remove crude debris masses. The supernatant fluid was loaded onto a column containing ECTEOLA-Cellulose and eluted with 0.07M phosphate buffer solution (pH 7.2). Some supernatant fluid was treated with fluorocarbon. In the case of yellow fever and Western equine encephalitis viruses, 100% of the original viruses was practically recovered and 90-93% of the nitrogen contained in the original virus materials was removed. Nitrogen contents of purified virus suspensions, as measured by the micro-Kjeldahl method, ranged mainly from 0.1 to 0.2mg/ml.

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A method is cescribed which shows that impurities contained in the chemically purified TMV preparations could be eliminated and two infective components could be separated by the use of strong cation exchange resin column (Amberlite IR-120).
The column (1×12cm) of the resin (Na-form, about 100 mesh size) was equilibrated with pH 5.0-5.2, M/15 phosphate buffer or M/25 citrate buffer solutions. Four to 8ml of TMV dissolved in the same buffer was added on the column. The column was then washed with 32ml of the same pH buffer, and 40ml of pH 6.2-6.3 and 40ml of pH 8.0-8.5 buffers were passed through the column. The impurities were contained in pH 5.0-5.2 fractions, and one infective component was obtained in pH 6.2-6.3 and the other in pH 8.0-8.5 fractions. The amount of the former component was much larger than that of the later.

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In the previous papers the author reported that Japanese B encephalitis and some other arbor viruses were partially purified by ECTEOLA-Cellulose column chromatography or fluorocarbon deproteinization.
The present paper describes that the partially purified viruses were treated with several enzymes and tested for sensitivity to the enzymes.
The enzymes used were trypsin, papain, bacterial proteinase, pancreatic lipase, fungal lipase, wheatgerm lipase, α-amylase, ribonuclease, and lysozyme. The enzymes, except for fungal lipase, were dissolved in 0.07M phosphate buffer solution (pH 7.2) at a final concentration of 0.125% and fungal lipase was diluted with 0.07M phosphate buffer solution (pH 6.0) to a final concentration of 125u/ml; one unit decomposes 1% of 2g of olive oil after exposure of 2.5 hours at pH 5.6 and temperature of 30°C. The partially purified viruses tested were Japanese B encephalitis, G1 strain; dengue fever type 1, Mochizuki strain; yellow fever, strain 17 D; Western equine encephalitis, Rockefeller Institute stock strain; and poliovirus, strain MEF-1. The viruses were mixed with enzymes, incubated at 37°C (except for fungal lipase which was incubated at 30°C) for 1 to 2 hours, and titrated for active virus contents by inoculation into either mice or tissue culture tubes. In controls, phosphate buffer solution was used in place of enzyme solution.
Results obtained are summarized as follows:
1) The infectivity of Japanese B encephalitis and other arbor viruses examined decreased markedly, or was lost completely, by trypsin or pancreatic lipase within 2 hours at 37°C. However, poliovirus was resistant to these enzymatic digestion.
2) Papain, bacterial proteinase, or fungal lipase also decreased (though less than trypsin or pancreatic lipase did) the infectivity of purified Japanese B encephalitis virus. On the other hand, wheat-germ lipase, lysozyme, α-amylase, and ribonuclease exerted little effect on the infectivity of the same virus.
3) When purified Japanese B encephalitis virus was treated with trypsin for 1 hour at 37°C and then exposed to ribonuclease for 2 hours, there was no reduction in viral infectivity.
It was finally concluded from the above results that trypsin and pancreatic lipase have an effect on substances controlling the infectivity of some arbor viruses. Although infective ribonucleic acid can be extracted from tissues infected with some arbor viruses, the intact virus particles are apparently resistant to ribonuclease. The various enzymesensitivities on the purified virus materials, in terms of the decrease of infectivity, are different from virus to another. Although the precise mechanism underlying the observed phenomena has remained unexplained, the enzymatic inactivation experiments may serve to analyse chemical structures of virus particles.

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