Two representative strains of Japanese encephalitis virus known to differ in the mouse pathogenicity, Nakayama-Yoken and JaTH-160, were investigated in more details for biological and biochemical properties as well as for morphology as seen under the electron microscope, using purified virus materials, and compared with each other to find out difference (s) which might account for the low and high pathogenicities for mice of these two strains. The results so far obtained indicate that the former strain possessing a low pathogenicity, as determined by the peripheral route infection, is covered with a rough surface envelope, on which are placed cylindrical and spike-like subunits 7-8mμ long with a central hollow in a radially manner; it showed an optimum pH 6.2-6.4 in hemagglutinin activity, and was more stable in the HA activity and infectivity than the latter. In contrast, the latter strain possessing a high pathogenicity has been found to be covered with a smooth envelope 6-8mμ thick; it showed an optimum pH of 6.6-6.8 in the HA activity, was less heat-stable in HA activity and infectivity and had a high ratio of CF titer/HA titer. Furthermore, a marked difference in the content of C₂₄ long-chain fatty acid was found in a lipid fraction of the latter strain, as compared with low contents thereof in Nakayama-Yoken strain. Serine, glycine and threonine contained in JaTH-160 was detected in higher amounts than in Nakayama-Yoken strain. However, contents of phenylalanine, lysine and histidine were very high in the latter strain. The buoyant density of Nakayama-Yoken strain was determined to be 1.34-1.35gm/cm³ in CsCl solution, while it was 1.30-1.31gm/cm³ with JaTH-160 strain. Those differences mentioned above between the two strains are discussed in relation to their different pathogenicities for mice as tested by the neural and extraneural routes.

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Epidemiological and virological studies of a large epidemic of echo-4 virus meningitis occurring in Japan in 1964 are described. The findings are as follows: (1) Out of about 400 specimens collected in the epidemic areas 152 strains of echo-4 virus were isolated by primary MK tube cultures. The isolation was successful up to day 9 after onset from spinal fluid (28.8% in the first week and 18.2% in the second week), day 16 from stools (61.4% in the first week and 44.1% in the second week), and day 3 from throat swabs. (2) The plaque method was ten times more sensitive than the tube method in echo-4 virus isolation from clinical materials and it afforded another advantage to have allowed quantitative measurement of existing virus in a material. The isolation rate from spinal fluid collected within 10 days after onset reached as high as 86% and that from blood taken within 5 days was 56%. The high rate of occurrence of echo-4 virus in spinal fluid and blood is considered to have significance in the pathogenesis of meningitis. (3) The current echo-4 virus isolated shared antigenic similarity, belonging to the Pesascek type which readily showed break through in neutralization. When compared serologically, they revealed rather unique antigenicity to the past echo-4 virus strains. (4) Human embryonic lung cell tube culture was of utility value for the neutralization of Pesascek type of echo-4 virus, since the endpoint was easily determined without break through. And also it was as useful as MK cells for virus isolation. (5) Seroepidemiological survey revealed that individuals under 9 years of age had no antibody to echo-4 virus before the epidemic in several areas of prevalence, thus it was concluded that the echo-4 virus epidemic broke out after an interval of about ten years. And other epidemiological features are described and disscussed.

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Various physico-chemical, serologic, and histopathologic properties of African horse-sickness (AHS) virus were studied using highly susceptible hosts, monkey kidney stable (MS) and African green monkey kidney (VERO) cell line cultures.
1. AHS virus was resistant to ether, trypsin, and heat, but was readily inactivated in acid.
2. Infectivity titers of AHS virus decreased markedly by storing infectious tissue culture fluids at temperatures between -20 C and -30 C. However, the infectivity was stable at -70 C. Inactivation of the virus was caused mainly by the salts used in virus suspensions. Infectivity of the virus was protected from inactivation by adding sugars (sucrose, lactose, or glucose) to the virus suspensions.
3. Complement-fixing titers of horse serums were 2- to 4-fold higher when the homologous antigens were prepared in MS cells. By improving this technique, complement fixation tests might be used for typing AHS virus.
4. There were no serologic cross reactions between AHS virus and arbo- and reoviruses.
5. Distribution of antigens in infected MS and VERO cell cultures was investigated by the fluorescent antibody technique. In the early stage of infection of MS cells, many granular antigens appeared in the cytoplasm. At a later stage, in many infected cells, 1 or 2 relatively large masses of antigens were observed in the cytoplasm and a fluorescent spot in or on top of the nucleus. Antigens were present in rosary-like cytoplasmic inclusion bodies dispersed along the nuclear membranes of infected VERO cells and appeared in the cytoplasm at the late stage of infection, in numerous granules.

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It was investigated whether tuberculin hypersensitivity could be induced in guinea pig embryos. Two fetuses in each litter have been injected with heat-killed tubercle bacilli in liquid paraffin through the intact abdominal wall of the dams about 7 days prior to parturition. Forty injections in all were performed in 20 dams. Eight out of 49 youngs delivered by the dams, excluding 16 stillborns, gave rise to positive tuberculin reactions at birth. Positive reactors increased to 20 animals when all the live young were skin-tested about 2 weeks after birth. The hemagglutinating antibody was also detected according to the Middlebrook-Dubos's method in all sera taken from 15 animals which showed the positive skin reaction, although the titers varied widely. These findings indicate that guinea pig embryos about 7 days before birth already have the capacity to produce both cellular and humoral antibodies.

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Guinea pig embryos 2 or 3 days before birth were thymectomized or sham-operated as soon as possible after the delivery of them by the cesarean operation. Several days after operation they were injected into foot pad with heat-killed BCG in saline. As a result both thymectomized and shamoperated animals similarly became tuberculin sensitive one or two weeks after the sensitization. Moreover, the circulating antibody formation as well as the gain of body weight were not affected by the thymectomy. On the other hand, peripheral blood leukocytes diminished slightly in the thymectomized group. Histological examinations of lymphoid organs at various stages of gestation revealed that the thymus and axillar lymph nodes were already lymphoidal in early fetal life. On the contrary, the spleen remained reticular in nature until a few days before birth. These findings indicate that the delayed type hypersensitivity is not affected by the thymectomy in the guinea pig embryos as well as the humoral antibody formation.

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Sodium ion and to a less extent potassium ion enhanced the growth and the syntheses of various toxic proteins of Clostridium perfringens PB6K. The optimum concentration of the total sodium and potassium ions was between 0.1 M and 0.15 M. Autolysis of the organism was often observed when grown in a synthetic medium suitable for toxin production. Calcium prevented the autolysis without affecting the yield of alpha toxin. Although autolysis was inhibited by most of other divalent cations, they were harmful for the toxin production. A new synthetic medium was formulated.

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When the washed toxoplasmas are used for the dye test high percentages of modified organisms are often produced in the accessory factor saline controls. The cause of such an increasing modification of washed organisms by an accessory factor serum was proved to be attributable to loss of heparin as a result of removal of peritoneal exudate in which the anticoagulant was involved. Addition of the conventional dose of heparin to the reaction mixture resulted in increasing stainability of the washed organisms in the negative control, but produced numerous fine floccules that rendered the microscopic reading of the stained and unstained organisms so difficult. The use of Alsever's preservative or 0.15 M citrate buffer solution at pH 5.3 gave clear-cut dye test results when they were added to the reaction mixture at a proportion of 6%. EDTA-Na₂ was also proved to be effective at a low concentration such as 0.043%, but in extremely narrow dose range. As the source of the accessory factor, plasma could be successfully used as in the case of using accessory factor serum plus Alsever. The favorable effect of the plasma was attributed to the presence of Alsever in it. A blindfold comparative dye test on 10 sera between the two laboratories, NIH of U. S. A. and NIH of Japan, resulted in excellent agreement. The favorable effects of these anticoagulants were thought to be attributable to chelation of excessive amounts of Mg and Ca, thus adjusting these cations to the moderate dose levels.

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