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Attempts were made to isolate Japanese encephalitis virus from mosquitoes collected with three light traps operated twice a week at three sentinel pigpens in the Taipei area from May to Oct. 1978. A total of 6,549 mosquitoes trapped alive were processed in 283 pools. Suspensions were inoculated into Aedes albopictus cell cultures and into suckling mice. A total of 19 JE virus isolates were made; 12 from pools of Cx. tritaeniorhynchus, 6 from Cx. annulus and one from Cx. quinquefaciatus. Sindbis virus was isolated for the first time in Taiwan from a pool of Cx. tritaeniorhynchus. More isolates were made from mosquito cell cultures (20) than by mouse inoculation (6). Positive isolations of JE virus by mouse inoculation were all from the mosquitoes obtained during an 11 day period from 20 to 30 June 1978 when all six sentinel pigs were viremic. This study shows the decided advantage of the use of mosquito-cell cultures over that of mice.

Japanese encephalitis virus was isolated from Culex tritaeniorhynchus, Culex bitaenorhynchus and Anopheles annularis mosquitoes collected from San Jose, Nueva Ecija, South Central Luzon in the Philippines. This is the second report of the isolation of the virus from mosquitoes in the Philippine Islands.

Ovarian cells from the mosquito Culex tritaeniorhynchus established in continuous tissue cultures have been found to support the growth of Japanese encephalitis virus with titers reaching as high as 10(7.36) MICLD50 per 0.03 ml. Virus-cell cultures were serially subcultured 60 times over 300 days and the virulence of the virus was determined after each passage. A gradual loss in virus titer was observed, with titers always higher in the medium than in the cells. Cytopathogenic effects were not observed in the infected cell cultures, either in primary or subsequent passages. However, the cell growth rate suggested that the cells may have been metabolically or mechanically damaged. The chromosome complement of the cells remained unchanged. Direct-fluorescent antibody studies during early and late passages revealed a loss in virus infectivity from chronically infected cells which may have been due to a gradual decrease in virus replication as the passages increased. The infectivity of virus particles, however, returned to previous levels after passage into fresh cell cultures. The loss of virus particles during serial passage may be correlated with the condition of the cells.

Sentinel pigs were bled and mosiquitoes were collected for isolation of Japanese encephalitis virus (JEV) between 1969 and 1973 in a rural area of northern Taiwan which reported a high annual incidence of human cases. The study site included a farmyard, schools, a bat cave, rice paddies and a heronry. Although Culex annulus was collected in every month of the year, isolations were made only in midsummer and only from Culex annulus and (once) from Culex tritaeniorhynchus. These isolates were usually collected from pig-related sources. Human cases in the vicinity of the study site were reported 3 to 6 weeks following the initial isolations from sentinel pigs. Isolations were made only for 4 to 8 weeks each summer. The isolation of JEV was not related to the availability of susceptible pigs or the spring or summer peaks in prevalence of Culex annulus. Despite periodic draining of rice paddies and the application of insecticides, sufficient numbers of Culex annulus were available to support transmission of JEV in each year of the study.

A total of 33, 274 mosquitoes was collected from Tagudin, Ilocos Sur Province, Philippines during July 1977. Isolates made from one pool of Culex tritaeniorhynchus and one pool of C. vishnui were identified as Japanese encephalitis virus (JEV) by a micro-neutralization test using reference hyperimmune mouse ascitic fluids. This is the first known isolation of JEV from the Philippines.

Female Aedes albopictus and Aedes togoi mosquitoes infected with Japanese encephalitis virus either by intrathoracic inoculation or by ingestion of a virus-sucrose-erythrocyte mixture transmitted the virus to a small percentage of their F1 progeny. Adult F1 female Aedes albopictus thus infected transmitted the virus in turn to newly hatched chickens by feeding on them.