An epidemic of rubella occurred in Toyama Prefecture in early spring in 1967. A prefecturewide hospital surveilance indicated that the epidemic was mainly localized in the south-westernpart of the prefecture, with Fukumitsu town as its center, that the peak age of the patients was 6 to 7, and that 18 of 22 patients over 20 years of age were female. Fever, whole body skin eruption, and swelling of the cervical lymph node appeared in 72.1%, 68.9%, and 41.6%, respectively, of the patients. Virological and seroepidemiological studies gave the following results. 1. Six strains of rubella virus were isolated from the throat swab and or blood of 5 patients consisting of 1 primary schoolchild, 3 junior-high-school boys, and 1 adult. 2. In patients with clinical manifestations, HI antibody response to rubella virus was rapid. The titer being 1:512 only 4 days after the onset of the disease. 3. After the epidemic, the antibody level was still very low (between ×8 and ×256) among nursery schoolchildren. It became higher with the advance in age. More than a half of the adults examined showed an intermediate antibody level (between ×8 and ×256). 4. Before the epidemic, 2 distinct patterns were observed in different districts with regard to the antibody level in various age groups. On antibody was detected from 2 age groups, 10 to 11 and 2 to 3. 5. The antibody level in adults after the epidemic was much higher than that before the outbreak. 6. Adults whose family member was infected with rubella virus showed a significantly higher peak in the distribution of antibody titers than those whose family member had not been infected with the virus. This may indicate the possibility that adults possessing a relatively low antibody level may have been reinfected with the virus or at least got a booster response to the virus.
Experiments were carried out on the plaque formation method of enteroviruses and in vitro marker tests of poliovirus. Comparison of the plaque technique and plaque-forming capacity was made between poliovirus type 1 and echovirus type 6 by using the Hela, FL, and monkey kidney (MK) cell culture systems. Intratypic specificity of antiserum was studied by the Wecker test. The temperature of the T-marker test and changes of T-marker were examined by passage of poliovirus type 1 strains on HeLa cells at 35°C. 1) The second agar overlay method was better than the single agar overlay method in the case of the plaque formation technique on continuous cell lines. 2) Plaque-forming capacity and infectivity titer of echovirus type 6 were greater on FL cells thann on MK cells. 3) The Wecker test used in the present studies was a slight modification of the original test (1960). RIST was calculated 4 days after inoculation. Isolates of poliovirus type 1 were differentiated in to sero-intratypic groups by it. 4) The T-marker test was carried out by a modification of the method reported by Yoshioka et al. (1959) and Carp et al. (1962). The test on HeLa cells was incubated in a water bath at 35 or 39°C (that on MK cells was incubated at 35 or 40°C & ±0.02°C) after 4 days of inoculation. Then plaques were counted. The results of the tests on both types of cells coincided in general. 5) The 1st, 2nd, 3rd, 5th, and 10th passages of the Mahoney, LSc. 2ab strains and isolates of poliovirus type 1 were incubated on HeLa cells at 35°C, but few of them changed T-marker. It seems reasonable to conclude from these tests that HeLa cells are applicable to the T-marker and Wecker tests, as well as MK cells.
There is a considerable amount of literature on T-marker and Wecker tests for poliovirus type 1 using monkey kidney cell cultures, but few experiments have been done to use a continuous cell line for these tests. In the present investigation, HeLa cells were used to examine the markers (T and Wecker's) of polioviruses and the relationship between these markers and their sources of isolation for epidemiological observation. 1) Of 9 wild strains isolated from patients with poliomyelitis, 8 strains were shown to be virulent and hetero-types (T+ or W+) and the remaining one was an intermediate type (T+ or W±). A strain isolated from a patient with aseptic meningitis was also T+ or W±. 2) Of 10 wild strains originated from healthy children, three were T+ or W+, four T+ or W±, and the remaining three T± or W±. 3) Of 16 isolates from healthy infants who had received oral live vaccine, fifteen were attenuated or homo-types (T- or W-) and the remaining one was T± or W-. The results mentioned above indicate that epidemiological observation is possible from the relationship between the markers of viruses and their sources of isolation.
Echovirus type 6 was isolated on FL cell culture from patients (infants and children) with aseptic meningitis, or so-called summer fever or diarrhea, and healthy children in Gifu Prefecture in epidemic stages during a period from 1961 to 1966. It was tested for various characters in FL and MK cell culture systems. 1) The recovery rate and the virus titer determined from the cytopathic effect of isolates on FL were higher on FL cells than on MK cells. 2) Non-passage virus (original echovirus type 6 specimens produced small plaques approximately 0.5 to 2.0mm in diameter predominantly on FL cells. About 0.5 to 1.0% of them were large-plaque-producing viruses. 3) Relative efficiency was higher in the large-plaque-producing viruses than in the small-plaque-producing viruses during passages of undiluted fluid specimens of stool suspensions, throat swabs, and cerebrospinal fluids on FL cells. 4) Isolates from the 2nd fluid passages on MK cells were tested for hemagglutinating capacity, but had no capacity of agglutinating human group O erythrocytes. 5) In neutralization tests, echovirus type 6 isolated on FL cells was divided into two groups, namely, strains neutralized by 10 and 100 units of anti (D'Amori) serum, respectively. In conclusion, no relationship was observed between any character of virus isolates and any symptom of patients from whom the virus had been recovered.
A total of 13 strains of Mycoplasma were isolated from 15 strains of established cell lines. All of them, except an unidentified strains were of human origin, including 5 strains of M. orale and 7 strains of M. hominis. Each of three cell strains, BHK21, RK13 contaminated with M. orale, and HmLu contaminated with M. hominis, was treated with a culture medium containing gentamicin (200γ/ml), kanamycin (250γ/ml), and tylosin (250γ/ml) or containing kanamycin and tylosin (250 or 500γ/ml each) for 9 days by changing the medium every other day. Mycoplasma was eliminated from RK13 cells even with the medium containing 250γ/ml each of kanamycin and tylosin and from HmLu cells only with the medium containing the three antibiotics. None of these cell lines yielded Mycoplasma during the subsequent 21 passages for a period of 150 days in the absence of the antibiotics used to the treatment. Mycolasma could not be eliminated from BHK21 cells, but the combination of the three antibiotics was more effective for treatment of the cells than that of two antibiotics. The results obtained suggest that the combined use of multiple antibiotics may be promising in eliminating Mycoplasma from cell culture, without giving any damage to the cells.
Studies were made on the physico-chemical properties of leucocytic pyrogens induced by influenza virus (V) and bacterial pyrogen of Escherichia coli (LPS) in rabbits. The following results were obtained. 1. The fever curve of the virus (256HAU/kg, i.v.) was quite different in lag time from that of LPS (0.1μg/kg, i.v.). The fever curves of the leucocytic pyrogens were quite similar to each other. 2. The chromatographic patterns over Sephadex (G-200) or Bio gel (P-150) of the two leucocytic pyrogens were shown to be the same as that of control leucocytic pyrogen. 3. The immuno-diffusion patterns of both leucocytic pyrogens against antiserum of guinea pig were the same, showing two or three precipitating bands. From the results mentioned above, it was concluted that both pyrogens (V and LPS) might be mediated by the same leucocytic pyrogens.
Experiments were carried out to examine the effect of the extract of the salivary gland of Myzus persicae Sulzer on broad bean epidermal cells infected with bean yellow mosaic virus (BYMV). The extract had been obtained by homogenizing the organ in distilled water. The extract of the salivary gland had an effect of producing microscopic changes in healthy and diseased epidermal cells. This effect was severer against virus-infected cells than non-infected cells. The most remarkable change in infected cells was the appearance of plasmolysis. This plasmolytic activity of the salivary gland extract was not destroyed by heat treatment. Virus inclusions in infected cells were granulated and diffused by the action of the extract. The cells plasmolyzed by the extract were different in shape from those plasmolyzed with 0.8M sucrose solution or 0 8M KNO3 solution. There was no significant difference in plasmolysis induced with the sucrose solution between healthy and diseased cells. The salivary gland extract was therefore presumed to act on virus-infected cells in a specific manner. The extracts of intestine, stomach, brain, and oesophageal ganglion caused mild plasmolysis of infected cells. They were, however, much weaker in plasmolytic activity than the salivary gland extract. Moreover, when exposed to this extract, the infected epidermal tissue decreased in virus activity. These results suggest that the saliva may play an important role on the transmission of the virus by the aphid; that is, by the influence of the saliva on infected cells, it may be difficult for the aphid stylet to acquire virus particles from these cells. The extent of the salivary effect would be determined by the interaction between the saliva and the plant cell. Therefore, the action of the saliva is presumed to be the cause of the low transmission of BYMV by aphid feeding.