71 PAPER IN SCIENTIFIC SESSION GROUP A: Immunology and Viral Diseases 1. Principles and Applications of Fluorescent Antibody Technique in Tropical Medicine Akira Kawamura Department of Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan 2. Studies on the Multiplication of Chkunya Virus (CHV), and on Physical and Chemical Structure of its Virion Noboru Higashi,Yasuko Nagatomo, Akira Tamura and Toshio Ametani Virus Institute, Kyoto University, Kyoto, Japan 3. Electronmicroscopic Studies on the Multiplications of Japanese Encephalitis Viurs Noboru Higashi*, Toshiko Ametani*, Yasuko Nagatomo*, Eiichi Fujiwara* and Takashi Tsuruhara** *Virus Institute, Kyoto Uniuersity, Kyoto, Japan and **NIH of Japan , Tokyo, Japan 4. Electromicroscopic Studies on the Multiplication of Dengue Virus Noboru Higashi*, Masao Tokuda*, Toshio Ametani*, Mya-Tu, My My Khin** and Toe Myint** *Virus Institute, Kyoto University, Kyoto, Japan and **Viral Section , Burma Medical Institute, Buruma 72 5. A Plaque Assay of Dengue and Other Arboviruses in Monolayer Cultures of BHK-21 Cells Hideo Aoki Department of Microbiology, School of Medicine Kobe University, Kobe, Japan A cell line of baby hamster kidney (BHK-21, clone 13) was found suitable for titration and multiplication of many kinds of arboviruses, including all types of dengue and related viruses. For instance, dengue (type 1, Hawaiian, Mochizuki ; type 2, New Guinea B ; type 3, H-87 ; type 4, H-241 ; type 5 ? Th-36 ; type 6 ? Th-Sman), JBE Nakayama, JaGar # 01, Gl) and Chikungunya (African) viruses are capable of producing clear plaques in monolayer cultures of the cells under a methyl cellulose overlay medium. By means of this plaque assay system, titration and neutralization of these arboviruses are possible. The plaque reduction tests can be made for titration of antibodies. Some basic aspects of arbovirus plaque formation have been studied. 6. Epidemiological Studies on the Transmission of Japanese Encephalitis. Results Obtained in 1968 Yoshito Wada Department of Medical Zoology, Nagasaki University School of Medicine, Nagasaki, Japan Epidemiological studies on the transmission of Japanese encephalitis have been conducted in cooperation with the Department of Virology, Institute for Tropical Medicine, Nagasaki University, in Nagasaki area since 1965. Mosquitoes were collected at regular or irregular intervals from early spring through late autumn in several villages around Nagasaki City, and Japanese encephalitis virus was iso- lated from those mosquitoes. Sera of slaughtered pigs were examined for HI antibody throughout a year. These data, together with the record of human encephalitis cases, were analysed. In 1968, the appearance of overwintered females of the vector mosquito, Culex tritaeniorhynchus, started on March 28, and that of newly emerged females started on May 14 ; the number of females was very small in March through May and small in June through August ; the first isolation of Japanese encephalitis virus was made on July 23 ; HI antibody possessing rate in slaughtered pigs reached 50 % in mid-August ; the peak appearance of human cases was seen in mid-September ; and the number of human cases was as small as 20. In comparison with the data obtained in the previous years, it can be said that the starting date for the appearance of overwintered females or of newly emerged females has no relation to the time of Japanese encephalitis epidemic in pigs or in men ; the appearance of infected mosquitoes and the rise of HI antibody possessing rate in slaughtered pigs are seen concurrently ; the size of human epidemic has a 73 positive relation to the number of vector mosquitoes in the period of the rise in HI antibody possessing rate of pigs. 7. Virologic-Epidemiological Investigations on Indonesia III. Comparative Surveys of Anti-Arboviral Antibodies in Sesa Collccted in Indonesia and Japan Susumu Hotta, Hideo Aoki, Tazuko Yasui and Susumu Samoto Department of Microbiology, School of Medicine, Kobe University, Kobe, Japan Sera were collected from residents of Indonesia, i. e., Lombok Island in 1964, Lampung State, South Sumatera, in 1965, and Madjalengka State, West Djawa, in 1966 ; as well as from those in Japan, in 1967. HI antibodies against dengue types 1, 2, 3, and 4 (D1, D2, D3 and D4), Japanese encephalitis (JE) , yellow fever (YF) and Chikungunya (CHK) viruses were measured using a microtiter technique (Takatsy-Sever). As to the Indonesian samples, it was indicated : (1) Antibodies against D1, D2, D3, D4 and JE were definitely found. Anti-CHK antibodies were also found, though in smaller percentages. (2) Quantitative distributions of the antibodies were different among the areas examined ; for instance, JE antibodies were com- paratively predominant in the 1964 Lombok sera, D1 and D4 in the 1965 Lampung sera, and D2 and D4 in the 1966 Madjalengka sera, respectively. It was presumed that each particular virus was predominantly distributed in the respective area. (3) Anti-YF antibodies were also detected in sera from all the areas surveyed. Because of their low titers, however, the YF-antibodies were presumed to be due to crossing reactions, especially by dengue infections. As to the Japanese samples, it was indicated : (1) Approximately 60% of the sera tested showed positive reactions against JE, or each type of dengue. No CHK-positive sera were noted. (2) Sera from individuals who suffered from den- gue during the 1942-44 Japanese epidemics showed positive reactions against dengue antigens, 20 years after what can be consibered to be a single infection. (3) Although there were sera possessing anti-YF atibodies, this was presumably due to crossing reactions by dengue. 8. Incidence of Bovine HI Positive Reactors to Japanese Encephalitis Virus in Indonesia Morimatsu Watanabe National Institute of Animal Health, Kodaira, Tokyo, Japan There are two possible hypothesis about the overwinter of the JEV One is 74 that the virus will hibernate in pig, lizard etc in Japan, and the other is that the JEV will over pass the winter time of Japan in southern warm countries. Japanese research workers showed the possibility of overwinter in lizard and still-born baby pig, while research workers in Okinawa and Formosa demonstrated the incidence of the Japanese encephalitis in pigs in the winter time of Japan. This paper shows the incidence of the disease in cattle, goat and sheep in Indonesia in 1967-1968 by means of serological tests. Methods Hemagglutination inhibition (HI) tests Hemagglutination (HA) antigen was prepared from suckling mouse brains infected with AS-6 strain of JEV by the method of acetone and ether extract of Clerk and Casals (1958). Test serum was treated with cold aceton and adsorbed goose erythrocytes for removing nonspecific inhibitors and isohemagglutinine. Using as diluent borate buffered saline (pH 9.0) with 0.4 % egg alubumin, serial 2-fold dilutions of test serum were mad in plastic tray and distributed in 0.2 ml amounts. To each well was added 0.2 ml of HA antigen containing 16 units. After incubation at 4 C for overnight, each well was received 0.4 ml of 0.33 % goose erythrocytes suspended in virus adjusting diluent (VAD) of pH 6.6. The test was read after further incubation at room temperature for 60 minutes. Neutralization tests Chick embryo fibro blast cells are used, grown in a medium consisting of Earle's balanced salt solution, 87.5 %; lactalbumine hydrolyzate, 0.5 %; calf serum, 10 %; 7.5 % NaHCO3, 2 % and antibiotics in solution. Serial two- or four-fold serum dilutions are mixed with an estimated 200 plaque-forming units of the JEV in Hank's solution containing 5 % antibody-free calf serum. The mixtures are incubated for 2 hours at 37 C and 0.2 % of each one is inoculated into two drained Petri dishes cultures. After an adsorption period of 90 minutes at 37 C, the mono- layers are covered with agar. The first overlay medium contains final concentra- tions of 1.0 agar, 0.5 % lactalbumine hydrolysate, 0.4 % glucose, 0.1 % yeast extract and 0.225 % sodium bicarbonate in Earle's solution. After 2 days of cultivation, a second overlay medium containing 1.0 % agar and 1: 10.000 neutral red in Earle's solution is added. Two days later the plaque counts of both dishes at each serum dilution are compared with a number of plaques in the control dishes without antiserum. The serum dilution causing 50 % reduction in plaque count are calcu- lated and expreses the neutralizing antibody titer. Results The results are shown in Table 1. In Timor island, positive HI reactions were found in 16 cattle among 87 examined, 8 goat among 64 examined and 5 sheeps among 42 examined. In Bali island, all cattle examined, which were harvested from so-called Djembrana disease, showed positive reaction. However, nothing can be said about the correlation between Japanese encephalitis and Djembrana disease. At present, at least it ban be siat that there are many positive HI reactors in animals in Indonesia in the winter time of Japan. 75 Table 1. Positive HI reactors in animals in Indonesia (1967-1968) Bovine sera were examined by neutralization tests to JEV and also showed posi- tive reaction. Concludion Incidence of Japanese encephalitis were found in Indonesia in cattle, goats and sheeps in winter time of Japan. These data may suggest that the JEV will over pass the winter time of Japan in warm countries and will come to Japan and cause the epidemics in Japan. 9. The Mode of Development of Japanese Encephalitis Virus in the Vector Mospuito as Observed by the Fluorescent Antibody Technique Rikuo Doi, Akiko Shirasaka and Manabu Sasa Department of Parasitology, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan A series of investigations have been carried out on the mode of development of Japanese encephalitis virus in mosquito organs at various time intervals from the infection, by application of direct fluorescent antibody technique on frozen sec- tions of the whole mosquito bodies.