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Several factors that were suspected of affecting the development of syncytia in cultured Aedes pseudoscutellaris cells inoculated with dengue viruses were studied. The results indicated that fresh media (less than 1 week old), low cell density at inoculation (2.8 × 105 cells/cm2), and low cell passage level (less than 52 passages) favored the development of syncytia. All three types (1, 2, and 3) of dengue viruses tested could be isolated from human sera by Aedes pseudoscutellaris cell culture by using syncytial development as an indicator, but the isolation rate was lower than that obtained by using intrathoracically inoculated Aedes aegypti mosquitoes.