Biomedical subjects
Publications and source records attributed to K Fukai.
Liposomes containing influenza virus hemagglutinin were reassembled from envelopes solubilized with Nonidet P-40 and were shown to induce hemolysis and cell fusion at low pH.
Application of the PAP technique for infectivity assay of mumps virus provides a fast, reproducible, and convenient assay system, which is better than other methods reported previously.
In Japan, dengue epidemics were recorded once before the end of World War II (1942-1945) on the Main Islands and several times (1893-1955) on the Okinawa Islands. Blood samples were obtained from residents in Osaka and Okinawa, and their antibodies were examined by neutralization tests against dengue and Japanese encephalitis viruses. Of 60 serum samples each from Osaka and Okinawa, 11 and 15 sera, respectively, showed positive titers against one or more dengue serotypes. These results confirm that the epidemics had been very large. Moreover, the results showed that the epidemics had been due to dengue type 1 virus in Osaka and to dengue type 1 and 2 viruses in Okinawa.
Two shots of the current Japanese encephalitis (JE) vaccine were given to children and their immune responses to the Nakayama strain (the vaccine strain) and two wild strains (JaGAr-01 and E-50) of JE virus were examined by neutralizing (N) antibody titrations. Seventy vaccinees had no N antibody to JE virus before the first vaccination and were bled one month after the second vaccination. The N antibody responses to the JaGAr-01 and E-50 strains were found to be similar and to be less than that to the Nakayama strain after the second vaccination: the geometric mean titers (GMT) of N antibodies to the JaGAr-01 and E-50 strains (as logarithms) were 1.87 and 1.75, respectively, while the GMT to the Nakayama strain was 2.89. The seroconversion rates to the Nakayama, JaGAr-01 and E-50 strains were 70/70 (100%), 69/70 (99%) and 68/70 (97%), respectively, after the second vaccination. Twenty-seven of the 70 vacciness were also bled before the second vaccination. Most of them showed a considerably high N antibody response against the Nakayama strain and only one vaccinee failed to show seroconversion after the first vaccination. However, the antibody response to the E-50 strain appeared to be rather low and 9 of 25 vaccinees did not show any seroconversion. Similarly 3 of 25 failed to show any seroconversion against the JaGAr-01 strain. These results indicate that at the initial immunization two shots, at least, of the current JE vaccine are necessary to stimulate effective immune responses to wild strains of JE virus.
Between September and November 1981, some members of a survey team from Japan suffered from a febrile illness diagnosed clinically as dengue fever during their stay in a village in Khon-Kaen Province, in the north-eastern part of Thailand. The morbidity rate in the team was as high as 69% (11/16). Blood samples were taken from 12 of the 16 members of the team in February, 1982 in Japan and the serum specimens were examined for antibodies to dengue (DEN), Japanese encephalitis (JE) and yellow fever (YF) viruses respectively. The results of the tests indicated that all 8 members who had had symptoms had been infected with DEN type 1 virus. No case of inapparent infection with dengue viruses was found. Of these 8 persons, seven had had neutralizing (N) antibody to JE virus before infection, but their clinical manifestations had been similar to those of an individual without N antibody to JE virus and were typical symptoms of dengue fever, such as leukopenia and "saddle-back" fever, without hemorrhagic manifestations, as seen from platelet counts and hematocrit values.
In 1943, a large dengue epidemic occurred in the Osaka district and several samples of dengue virus were isolated from patients with dengue fever by workers in this Institute. These were inoculated into human volunteers to confirm that they were dengue virus. In the present study, serum samples were collected from the volunteers who had been inoculated with dengue virus and were examined serologically. In the neutralization test, all the sera showed a higher titer against dengue type 1 virus (DEN-1) than against the other three types of dengue virus, indicating that the virus strain isolated in 1943 was DEN-1.
Two shots of the current Japanese encephalitis (JE) vaccine were given to children and their immune responses to the Nakayama strain (the vaccine strain) and two wild strains (JaGAr-01 and E-50) of virus were examined by neutralizing (N) antibody titrations. Seventy vaccinees without antibody to JE virus before the first vaccination were bled one month after the second vaccination. The N antibody responses to the JaGAr-01 and E-50 strains were found to be similar and to be less than that to the Nakayama strain after the second vaccination: the geometric mean titers (GMT) of N antibodies to the JaGAr-01 and E-50 strains (as logarithm) were 1.87 and 1.75, respectively, while the GMT to the Nakayama strain was 2.89. The sero-conversion rates to the Nakayama, JaGAr-01 and E-50 strains were 70/70 (100%), 69/70 (99%) and 68/70 (97%), respectively, after the second vaccination. Twenty-seven of the 70 vaccinees were also bled before the second vaccination. Most of them showed a considerably high N antibody response to the Nakayama strain and only one vaccinee failed to show seroconversion after the first vaccination. However, the antibody response to the E-50 strain appeared to be rather low and 9 of 25 vaccinees did not show any seroconversion. Similarly 3 of 25 failed to show any seroconversion against the JaGAr-01 strain. These results indicate that at the initial immunization at least two shots of the current JE vaccine are necessary to stimulate effective immune responses to wild strains of the JE virus.
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One of the authors (Y.O.), who had previously been immunized with Japanese encephalitis (JE) vaccine, showed symptoms of typical dengue fever 6 days after accidental infection with a newly isolated dengue type 4 virus strain from a patient with dengue hemorrhagic fever (DHF) in Thailand. His sera were examined by hemagglutination inhibition (HI), complement fixation (CF) and neutralization (N) tests. The JE N antibody titers of his sera were high even on the first day of the illness and remained almost constant during the next year. Antibodies that reacted with dengue viruses were detected from a very early stage of the illness by all three serological tests. In addition, his convalescent phase sera showed high titers against all 4 types of dengue virus. These data suggest that the dengue infection caused secondary stimulation of antigens of flavivirus. Sedimentation analysis of antibodies in Y.O.'s serum (day 9) was carried out and IgM antibody that reacted only with dengue type 4 virus and homologous infecting virus was separated. These findings clearly demonstrated that the laboratory infection of Y.O. was primary dengue infection with dengue type 4 virus.
Influenza virus hemolytic activity was found to be more heat-resistant than neuraminidase activity, and to be heat-inactivated similarly to hemagglutination activity, in sharp contrast to the case with paramyxoviruses, where the hemolytic activity is the most heat-labile, and hemagglutination and neuraminidase activities are inactivated similarly by heat. Influenza viruses with heat-inactivated neuraminidase activity, which still showed hemagglutination and hemolytic activities, were found to be able to induce cell fusion and envelope fusion. This finding suggested that the hemolytic and fusion activities are not dependent on neuraminidase activity. The hemolytic activity was largely inhibited by anti-hemagglutinin serum of the same subtype. The pH range, heat stability, and antiserum susceptibility of the hemolytic activity were found to be independent of the cells in which the virus was grown.
One of the authors (Y.O.) of this paper was accidentally infected in the laboratory with dengue type 4 virus, isolated in Thailand in 1978 using the C6/36 clone of Singh's Aedes albopictus cells (SA). Two strains of the virus were isolated from Y.O.'s serum on the first day of illness using SA and suckling mouse brain (SMB). The SA-isolate and the infecting virus were neutralized with high efficiency by Y.O.'s convalescent sera, but the SMB-isolate was neutralized much less efficiently by the same sera. When the hosts of the isolates were exchanged (SA to SMB, and vice versa), the neutralization efficiencies were reversed. For analysis of this phenomenon, prototype dengue type 4 virus exclusively passed in SMB and the same type of virus passaged 10 times in SA were compared with the initial SA- and SMB-isolates in neutralization tests with sera from patients with dengue hemorrhagic fever (DHF) obtained in Thailand. The two strains of the prototype virus and the SA-isolate had similar high reactivities, but the SMB-isolate had low reactivity with sera of the DHF patients.
The role of pontine reticula units (PRU) responding to afferent impulses from the distal colon and anal mucosa was studied in dogs. The 465 PRU were classified into 4 groups on the basis of the pattern of discharge in response to afferent stimulation (AS) of rectal branches (RB) of the pelvic nerve: Group I (G-I) responded with a short burst (latency, 36 +/- 16 msec); G-II responded with an early short burst (latency, 41 +/- 21 msec), followed by a late long-lasting train of spikes (latency, 187 +/- 110 msec); and G-III responded with a long-lasting train of spikes (latency, 189 +/- 130 msec). G-IV had spontaneous discharges which were temporally inhibited by AS. G-II and G-III PRU showed remarkable facilitation similar to that of the long latency reflex discharges of RB. G-I PRU showed habituation. Most of G-II and G-III PRU discharged simultaneously with the reflex discharges of RB; these cause the contractions of defecation in the distal colon. In each group, 6.4 +/- 14.8% of PRU were reticulospinal units with descending axons running beyond the 1st lumbar segment of the spinal cord. It was concluded that the pontine defecation reflex center plays an essential role in defecating activity.
The early interactions of LLC-MK2 cell-grown noninfectious Sendai virus and a murine cell line, P815 mastocytoma ascitic cells, were studied by electron microscopy, using the ferritin-conjugated antibody technique with anti-virus glycoprotein serum. For comparison, the interactions of egg-grown infectious Sendai virus with the same cells were also examined. When noninfectious virus was adsorbed to the cells in the cold, the cell membranes become partially invaginated at the site of contact of adsorbed virions, but ferritin-conjugated antibodies did not penetrate into the areas of envelope-cell membrane association. This pattern of virus attachment was similar to that of infectious virus attachment. Upon subsequent incubation at 37 degrees C, most of the adsorbed noninfectious virions were taken into cytoplasmic vesicles and then degraded, although a few virions remained attached to the cell membrane. No evidence of fusion of envelopes of noninfectious virions was obtained. On the other hand, envelopes of infectious virions fused with the cell membrane, and the transferred viral antigens diffused on the cell surfaces and then decreased in number.
Isolation of viruses from female Culex tritaeniorhynchus was performed by inoculation of specimens into Aedes albopictus clone C6/36 cells as well as into suckling mouse brains (SMB). Altogether, 32,812 mosquitoes in 349 pools were processed from the specimens collected in Osaka Prefecture during the summer of 1978. Thirty-nine strains of Japanese encephalitis virus (JEV) were isolated in C6/36 cells in contrast to 27 strains in SMB. Twenty-four pools yielded JEV both in cell cultures and in SMB, 10 yielded JEV only in cell cultures, and three only in SMB. Two strains of Getah (GET) virus were isolated in cell cultures but not in SMB. Two of the 23 plaque isolates of a GET strain showed significantly lower titers of plaque formation on BHK21 cells or SMB-LD50, compared with the titer of plaque formation on C6/36 cells at 28 degree C. In addition, many unidentified filterable agents were detected by plaque formation on C6/36 cells.
Single doses of Japanese encephalitis (JE) vaccine of the Nakayama strain were given to members of the staffs of a hospital and 99 paired-sera were obtained. Neutralization antibody titers of the sera were determined against the Nakayama strain and E-50 strain, isolated from wild Culex tritaeniorhynchus using clone C6/36 of Singh's Aedes albopictus mosquito cell culture. Post-vaccination sera showed a significant neutralizing antibody response against both the Nakayama and E-50 strain. The geometrical mean neutralization titers of post-vaccination sera against the Nakayama and E-50 strains were in the same order, although the geometrical mean neutralization titer against the Nakayama strain was the higher in pre-vaccination sera. The antibody responses against the Nakayama strain and E-50 strain were somewhat different in detail: 7 individuals with a pre-vaccination neutralization titers of less than 10 showed antibody responses to the Nakayama strain, whereas 3 individuals of the same group did not show any detectable response to the E-50 strain.
Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.