The virulent Towata strain of mumps virus was successfully passaged in the chorio allantoic cavity of developing chick embryos, and it was noticed that Towata strain was considerably attenuated after many passages (described in the first report). Attenuated Towata strain of various passage numbers was used in 4 field trials for children in 1967 by the inhalation method. Similar degree of antibody response was obtained in each trial, without any clinical manifestations due to the vaccination. Increase in NT antibody titer was 8 to 18 fold, and 80 per cent of seroconversion was observed in sero-negative cases. The rate of sero conversion of NT antibody was higher than that of HI antibody in paired sera. It was noticed that many children who had low antibody also showed antibody response. In inhalation trials with Enders strain adapted to the chorio allantoic cavity, antibody response almost similar to that of Towata strain was obtained.
An African green monkey kidney cell line, Vero was tested on its viral susceptibility to 81 laboratory strains covering Paramyxo. Influenza, Entero, Reo, Arbo, Adeno, Herpes, Papova and Poxvirus groups. A wide range of viral susceptibilities was demonstrated excepting the followings·Influenza A2 and B, and among Paramyxoviruses, HA-2, Parainfluenza type 4 and one of the two strains tested of HA-1 was nonpassable or of incomplete reproduction cycle. Coxsackie A-9 virus showed a low titer in this system. Some types of ECHO virus and one of Japanese encephalitis virus (Mukai-m) gave suggestive results for low susceptibility of this cell line. To all other virus strains tested Vero showed good sensitivity as can be used for infectivity titration and virus neutralization tests. On the chromosome analysis Vero showed several characteristics of pseudodiploid nature and cercopithecus origin.
The present report deals with the production and characterization of inactivated Japanese encephalitis virus vaccine grown in porcine kidney cells for human trials. Bottle cultures of RFVL line of Nakayama strain were completely harvested by freeze-thawing followed by sonication. This improved the potency of the vaccine although there was no apparent rise in virus titer. The paradox may in part be explained by the lability to the temperature of the virus. In fact, the halflife of the virus was measured as follows: 15hr at 30°, 11hr at 33°, 7hr at 36° and 5hr at 37°. The resulting virus suspension was further centrifuged at 10, 000×g for 30min and filtered through membrane filter of 0.45μ pore size with little loss of virus. As inactivating reagent, formalin was superior to β-propiolactione since the latter significantly impaired the antigenicity. Optimal concentration of formalin was found to be 1: 2000 to retain the immunizing potency. However it must be kept in mind that over-neutralization by sodium metasulfate of formalin may be detrimental to the antigenicity. Safety tests were performed at appropriate stages of processing; these included sterility test, safety test in a narrow sense, and exclusion of recidual JEV as well as contaminating or adventitious agents in porcine kidney cells. Potency test was carried out in mice in two ways; one was the direct challenge method required by the Japanese government, and the other was to evaluate the antigenic extinction limit method as determined by the 50% plaque reduction. In our experience the latter technique was bettter in that it was more sensitive and reliable. The potency of the tissue culture vaccine was estimated to be 1/2 to 1/4 of that of the mouse brain type of vaccine. The stability of the vaccine was observed by storing it at refrigerator temperature for 3 years, during which period the relative potency of the tissue culture vaccine to current mouse brain vaccine remained unchanged. Finally the vaccine was tested in both man and monkey for antibody response. It was revealed that the tissue culture vaccine was not satisfactory in a primary response but showed definite booster effects. No untoward reactions were noted among vaccinees.
Mixed infection experiments of vaccinia subgroup viruses were done using plaque types and characters of A type inclusion as genetic markers. It revealed that A type inclusion marker could be divided into two parts; formation of inclusion matrix (In+ and In-) and virus attaching factor (V+ and V-). Recombinant between V and In markers was not found in over 200 progeny clones of mixed infection, but recombinants between plaque types and A type inclusion types were detected. Vaccinia IHD, which does not form A type inclusion by single infection, can rescue all V- type inclusion in mixedly infected cells and it results in formation of V+ type inclusion.
Propriety of immunofluorescent procedures for the cells on a cover slip obtained from the VSV infected L cells in suspension was examined in qualitative and quantitative manner. The cell number could be quantitatively recovered from the whole cover slip when transfered from the mother culture. The fixation, staining and washing procedures as used routinely in immunofluorescent study had no influence on cell number of the cover slip. Although the distribution of cell number varied from one microscopic field to another, there was no difference in proportion of the infected to the uninfected cells by means of the immunofluorescent staining. These observations may give a warrant for the usual cover slip method, where only a portion of the cells in the mother culture is to be examined, that the cell population on the whole cover slip can be a representative of the mother culture.
Japanese B Encephalitis viruses (Furumoto and Nakayama strain) were inoculated into the colostrum-deprived new-born pigs and their clinical symptoms, viremical patterns, virus infective titers in the various organs, localization pattern of the viral antigen and the mode of antibody formation were studied. 1. As clinical symptoms, high fever, anorexia and general malaise were mostly observed. Convulsions and tremors were also observed in the Furumoto-inoculated cases. A transient paresis of the hind legs was observed in some of the Nakayama-inoculated pigs. 2. Viremia occurred in all cases but was most obvious in the case of fresh Furumoto strain and continued for 96 hours. 3. Virological observations revealed the high concentration of infective viruses in the brain, lymph node and lungs, but the date of maximum. concentration was different in each organ. 4. Immunofluorescent technique was adopted to check the localizing pattern of Japanese B Encephalitis viral antigen in the tissues. It reveals that the viral antigen seemed to multiply themselves in the reticuloendothelial cells even on the first day after inoculation, and caused viremia. Some viral antigen was seen in the bronchial epithels, too. After viremia, a generalized inflammation occurred and the viral antigens were observed in macrophages and large mononuclear cells. In the nervous system, viral antigen appeared temporally in the peripheral nerve sheath of some animals. In the central nervous system the viral antigen generally did not pass through the blood brain barrier, However, in the animals which showed a paresis of hind legs the viral antigen appeared in the ganglion cells of basal nuclei on the same day as the paresis occurred. 5. The infected colostrum-deprived pigs started to produce antibody (hemmagglutination inhibition antibody) on the 4th day after inoculation, when the circulating antigen disappeared rapidly. The viral antigen in the tissue disappeared on the 7th day when the amounts of circulating antibody came to its maximum.