Plaque formation by the HF strain of herpes simplex virus in HeLa cells under agar overlay was possible when a line of this strain highly adapted to HeLa cells was used, whereas the homologous strain, passaged in eggs or lowly adapted to HeLa cells did not form any visible plaques using the same technique. Experiments were performed to investigate the influence of different diluents, cellular factors and conditions of virus adsorption. It was found that 0.1% yolk-saline could replace the ordinarily used main-tenance medium as a diluent. Among three lines of S3 HeLa cells maintained indifferent laboratories, a subclone possessing a rapid cellular growth rate showed the highest sensitivity to the virus. Monolayers which scarcely covered the whole glass surface were most suitable for virus inoculation. Such monolayers could be stored at 20C for 2 days before use without lowering the efficiency of plaque formation. The highest titer of virus was obtained when adsorption of inoculated virus was allowed to take place at 37C for 2hr. This titer was about equal to the plaque titer obtained in primary chick embryo cell cultures.

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An F + strain of E. coli K 12, W-2241 has a genetic constitution lac^- (i⁺ z⁺ y^-), str-r. The inability of this strain to utilize lactose is due to a deletion at the y locus con-trolling formation of an enzyme permease and therefore, a true lac⁺ revertant can not be expected. The strain, however, produces many colonies on minimal lactose agar medium, when it is plated after infection with RNA phage Qβ, while much fewer colonies are observed on the same medium seeded with uninfected cells. In analyzing this phenomenon, we discovered that these LAC⁺ colonies were not the result of a mechanism similar to transduction but produced by a mechanism related to phage liberation, and that the colonies originated from Qβ⁺, const cells. The data suggest the following working hypothesis for the mechanism by which LAC⁺ colonies are produced in a population of W-2241 cells infected with phage Qβ. Constitutive cells carrying Qβ produce intracellular β-galactosidase and at the stage of phage liberation, the enzyme is liberated into the medium, hydrolyzing lactose to form glucose. Since glucose could be utilized by all of the cells on the medium, the Qβ+, const cell acts as a colony forming center. The phenomenon described in this paper shows a new type of virus-host relationship which may have some bearing on the influence of animal or plant viruses on host tissues.

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An epidemic of pharyngoconjunctival fever due to adenovirus type 3 occurred among children in a primary school in Japan during summer months of 1964. The etiologic diagnosis of the epidemic was readily made by virus isolation and serologic tests on patients. Adenovirus type 3 was isolated in HeLa cell cultures from 55% of 52 patients, and serologic evidence for type 3 virus infection was obtained in 93% of 54 patients which were tested for complement-fixing, neutralizing, and hemagglutination-inhibiting antibodies. The HI test was found to be as useful in the serologic diagnosis of the serotype as the NT test. It is evident that for the routine use, the HI test is more advantageous than NT test as the latter is more laborious and time consuming. The fact that a strain recovered in this epidemic gave higher HI titers than the prototype strain, when used as the antigen in HI tests with sera of patients in this epidemic indicates the desirability of selecting an appropriate virus strain for preparation of the antigen for HI test in serological diagnosis of patients.

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Thirty-seven strains of parainfluenza 3 virus were isolated on bovine kidney cell cultures from Japanese cattle in herds suffering from acute respiratory illness. This finding, together with the clinical and epidemiologic observations and the results of a serologic survey, indicates that in Japan, as in the United States and other countries, this virus is one of the important causes of acute respiratory disease in cattle. A significant finding in the present study is that virus was recovered from milk as well as from nasal secretions. This finding suggests an important role for milk, along with nasal discharges, in disseminating the virus among cattle. In addition, the recovery of virus from milk presents a new problem concerning this infection in cattle, in particu-lar, the potential role of this virus in the pathogenesis of mastitis.

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In the previous study, parainfluenza 3 virus was isolated not only from nasal secretions but also from the milk of naturally infected cows. In the present study it was confirmed that the virus isolated by us is similar in its pathogenicity for cattle as the bovine strains of this virus previously reported. In view of the fact that the virus was isolated from milk, the virus was inoculated directly into the udders of lactating cows. The infected cows showed the same respiratory symptoms as well as the symptoms of fever, malaise, anorexia, diarrhea and leukopenia as observed in cattle infected by other routes of inoculation. The affected udders developed swelling and induration, and milk from these glands showed coagulation, color change, and increased pH and cell count. The milk contained increased numbers of glandular epithelial cells, mature and young neutrophils and monocytes. Virus was excreted in large amounts in milk from the quarters inoculated with virus. However, many of the uninoculated quarters also excreted virus in milk, but in smaller amounts. Virus was recovered from blood and nasal discharges of infected cows. Antibodies were produced in the infected cows and were excreted in milk. These findings, together with the clinical observations and the results of virus isolation from milk in natural cases, indicate that, in natural infection of cows with this virus, udder infection occurs frequently, but seldom results in overt clinical mastitis. However, the role and significance of the virus in the pathogenesis of mastitis should be further investigated.

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Propagation of Japanese encephalitis (JE) virus in cells of dog cerebellar tissue cultures was investigated by means of fluorescent antibody (FA) technique. The fluorescent globulin conjugate was made from the serum of a dog inoculated with partially purified JE virus, treated by Sephadex G-25 and DEAF cellulose column chromatography and then adsorbed with dog liver powder. This preparation was found to be appropriate for the present work. Fluorescence was demonstrable in virus-infected cultures of three different types of cells, fibroblast-like cells, nerve cells and some of the glial type cells. Fluorescence could first be demonstrated about 20 hours after virus inoculation and appeared to increase in intensity in proportion to the increase of infective virus present in the cultures. The specificity of the reaction was supported by the non-reactivity of control (non-infected) cultures and by the results of blocking tests. The infected nerve cells and glial type cells also exhibited morphological changes clearly detectable by the FA techniques, corresponding to the changes shown in Bodian-stained preparations. The localization of FA antigen in the fibers of these cells suggests a possible mode of spread of JE virus in the nervous tissues. In any of the cell types studied thus far, the nuclei remained FA-unstained even during the advanced stage of infection.

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