Comparison of three methods used to isolate dengue virus type 2.

During the 1969 dengue epidemic in Puerto Rico, human sera and Aedes aegypti mosquitoes were collected for virus isolation and identification. Three methods of isolation were used and compared. In the first method, we inoculated newborn mice by the intracranial route, noted any signs of illness, and serially passed specimens in mice until virus was isolated. In the second method, we inoculated tube cultures of LLC-MK(2) cells, noted any cytopathic effect (CPE), and assayed fluids for virus by plaque formation in LLC-MK(2) cell monolayers. The third method was different from the second only in that the original specimens were first inoculated into fluid cultures of Singh's A. albopictus cells. No significant CPE was seen in LLC-MK(2) cultures; however, distinct syncytial CPE was observed in A. albopictus cells. About the same number of virus isolates were made in each isolation system. Virus isolates from both sera and mosquitoes were identified as dengue type 2 by a plaque-reduction neutralization test in LLC-MK(2) cells. The utility of the three methods, individually or in combination, is discussed and related to diagnostic and epidemic situations.