Three strains of rubella virus could be isolated with primary African green monkey kidney (GMK) cell from the throat swab specimens of rubella patients during the epidemic at Osaka district in the spring of 1966. One of these newly isolated strains has been serially cultivated for at least 11 passages in the amniotic cavity of a developing chick embryo.
The chicken egg adapted strain was rather sensitive to temperatures as compared with other strain which was passaged only in GMK cell several times after isolation.
It was experimentally inoculated into cynomolgus monkeys at the 6th and 11th passage level and both produced sufficiently high NT (neutralization) and HI (hemagglutination-inhibition) antibody responses without any recognizable symptoms, although there were a little virus recoveries from the throat of some monkeys at both passage levels.

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Cytopathic effect in Vero cultures caused by the two representative laboratory strains of rubella virus was investigated from the point of view whether the CPE is virus specific or not. Serologically, the CPE was neutralized by rubella convalescent patients' sera to high titers whereas no neutralization was observed by various kinds of heterologous virus antisera. Rabbit immune sera prepared in this laboratory against the Vero-grown rubella viruses did not neutralize heterologous viruses covering various groups except HVJ against which the preimmunization rabbit sera were found to be neutralizing, too. Hemagglutinin against goose erythrocytes was produced in the infected Vero cultures. Using this hemagglutinin hemagglutinin-inhibition tests were performed on the rubella patients' paired sera, anti-rubella virus sera obtained from other laboratories and the above-mentioned homologous and heterologous immune sera in parallel tests with BHK-21 produced hemagglutinin. Comparable results were obtained with both HA antigens, so that specificity of the hemagglutinin produced in Vero was acceptable.
Neither cytoplasmic nor intranuclear inclusion bodies were found in the hematoxylin-eosin stained preparations of infected Vero cells. No hemadsorption of guinea pig erythrocytes was observed in the infected cultures. No production of hemagglutinin for chicken red blood cells in allantoic fluid occurred after allantoic inoculation of embryonated chicken eggs with the Vero grown viruses. New born mice survived without showing any symptoms after intracerebral inoculation with the agent. Sensitivity to chloroform and RNA type of the agent as determined by IUDR inhibition test of virus growth were also proved. Filtration tests using Millipore membranes gave suggestive results of around 50mμ diameter of particle size of the CPE agents. Rapid inactivation by 1:4000 formalin at 37°C was demonstrated with the Vero grown viruses as tested in Vero cultures for residual infectivity.
These results do not contradict the reported properties of rubella virus and can exclude a wide range of heterologous viruses as the CPE-causing agent for Vero cells or as contaminating viruses in the virus stocks tested by us. If follows that the cell line, Vero, can be used as one of the suitable CPE system for rubella virus study.

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A strain of SV 41 given by R. M. Chanock was grown and passaged in Vero cells (Cercopithecus kidney cell line). In the reciprocal cross neutralization tests among Paramyxoviruses SV 41 was clearly distinguished from other viruses except that slight crossing was observed against CA virus. Some physico-chemical tests, including size determination by filtration, sensitivity to chloroform and nucleic acid typing using IUDR, and some host range tests gave results essentially consistent with those described originally by Miller et al. on primary culture-grown viruses. Contrary to our expectation in the heating tests at 50°C SV 41 was not labile; that is, after heating for 9 hours a decline of infectivity by 5 logs was demonstrated but still left 1.5 log of infectivity.
Search for serum neutralizing antibodies in humans, monkeys and various experimental and domestic animals revealed that the sole species which gave positive results was humans and no positives were encountered in 321 monkeys including Cynomolgus, Rhesus and Cercopithecus species which were imported from 1960 to 1966 and kept for varying periods of time in this laboratory, Whether the SV 41 antibodies in humans are due to infections of SV 41 itself or not can not be decided from our present data. Its confirmation waits for future investigations.

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