(1) From ultraviolet inactivation curves, RNA phages used in this study were separated in three groups (I:II:III=1:1.4:2). The result is in good agreement with a serological and a physicochemical criteria. (2) The bacterial capacity to support the reproduction of RNA phages were reserved well in heavily ultraviolet inactivated cells as in nonirradiated cells. (3) No evidence of host cell reactivation and ultraviolet reactivation was found in ultraviolet irradiated RNA phages.
We have succeeded in isolating 34 strains of RNA phage from the sources collected in various parts of Japan. We have also grouped these strains and several known RNA phage strains (MS2, f2, R17, Qβ) serologically, and discovered that these RNA phages were separated in 3 groups. The frequencies of the phages of these 3 groups in natural environment were almost equal. So far, none of RNA phage have been discovered, that do not belong to any of these 3 groups. The results of serological grouping were confirmed also by a Millipore filtration method, UV sensitivity, and some other physicochemical methods.
The present report concerns the multiplication of the Japanese encephalitis virus in primary porcine kidney cell cultures. Suckling and weanling pigs were constantly excellent donors of tissues. Viability of trypsinized cells was dependent on washing procedure; this was especially the case with tissues from marketable animals. Confluent sheets were obtained in 5 to 7 days, and the virus was usually inoculated on the 7th to 8th day of incubation. Unfortunately, medium 199 did not support the growth of viruses so well as did the Lactalbumin hydrolysate-Earle's medium. So far tested LE medium fortified with bovine albumin was the best one, although this could be substituted by human albumin or gelatin. The optimal concentration of sodium bicarbonate fell in between 2.5 and 2.6g per liter of medium. At the early stage of adaptation the viruses were sensitive to the lower temperature, while adapted strains were not affected appreciably by the temperature. In order to have a maximal yield relatively large size of inoculum was required, the optimum being 104 to 105 PFU per milliliter of medium. Growth curves showed peak titers at 48 hour of incubation both in cells and fluid phase. Some promissing results were also obtained by submerged cultures.
Pathology of the experimental Japanese encephalitis (JE) has been investigated using various laboratory animals. In this report, only the pathological examination of rhesus or cynomolgus monkeys weighing 2 to 3kg. is summerized. Used virus strains of JE were Nakayama-NIH and JaTH160 and 0.1 to 0.2ml. or 0.5ml. of virus suspension containing 102.6 to 106.9 BLU (50% suckling mouse lethal unit) were injected intracerebrally or subcutaneously. After inoculation, the symptom, body temperature, and body weight of the infected monkeys were checked every day, and the dead or sacrificed animals in agonal stage were bled for serological examination. Finally autopsy was performed after 10% formalin perfusion. The specimens from each area of the central nervous system (CNS) were frozen by dry ice aceton for fluorescent antibody technique (FAT) assay and virus titration. The peripheral injection with 106 BLU of JaTH160 to 6 monkeys showed inappearent infection. However, the pathological findings regard as the visceral phase previously described by others were not clear, but swelling of the germinal center in the lymphoid tissue was observed, though it could not been decided whether it was the specific change induced by virus infection or not. On the other hand, the intracerebral inoculation revealed the typical encephalitis symptom, and exhibited histologically focal character of the lesion and distribution of foci similar to other animal infection. Neuropathologically, marked changes were observed in the spinal cord, especially, in the anterior horn as if they are observed in Heine-Medin's disease. Therefore, so long as the histopathological changes of the spinal cord is concerned, differential diagnosis between JE and poliomyelitis appeared very difficult. The distribution of foci was observed along the ventricular system as if there is a correlation between the histologic changes of the spinal cord and flow of cerebrospinal fluid (CSF). On the FAT assay, the distribution of viral antigen was exclusively observed in the nerve cell, and no viral antigen was found in the other tissues and cells. These results corresponded with histopathological appearance, but it was infered as FAT findings was rather antecedent to histological appearance. The virus content in each area of CNS was investigated with suckling mice. In early stage of the inoculation, the brain stem centered in the middle brain showed high viral titer. With progressing of the symptom, the spinal cord, especially the lumbal cord, showed the highest titer. These findings coincided with histological appearance. However, the cerebellum showed the high infective titer in spite of its scanty histological change, and CSF and the serum showed so low titer being against one's expectation. The minimal infectious dose of JE virus to monkey was assumed as 102 BLU, although the intensity of pathological changes were not pararell with viral dosis.