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  • Title: Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. I. Attributes of uncloned virus at different passage levels.
    Author: Halstead SB, Diwan AR, Marchette NJ, Palumbo NE, Srisukonth L.
    Journal: Am J Trop Med Hyg; 1984 Jul; 33(4):654-65. PubMed ID: 6476212.
    Abstract:
    Attempts were made to attenuate prototype dengue (DEN) 4 (H-241) virus. The original viremic human serum was passed once in a susceptible monkey and twice in Aedes albopictus mosquitoes and then serially passed in primary dog kidney (PDK) and African green monkey kidney (GMK) cells. Weekly transfers of undiluted virus were carried to the 50th passage in both primary cell cultures. Biological markers were studied at passages 7, 15, 30 and 50. Parental DEN-4 phenotype characteristics included large plaque formation in LLC-MK2 cells, plaque formation in GMK cells, cytopathic effect in LLC-MK2 cells, growth in human monocyte cultures, growth at 39 degrees C, consistent production of viremia in monkeys and short-incubation neurovirulence in mice. At the seventh passage in both PDK and GMK cell cultures, DEN-4 viruses exhibited reduced plaque-size in LLC-MK2, and failed to plaque in GMK, to produce cytopathic effect in LLC-MK2, or to grow in human monocytes. Serial passage in PDK, as opposed to GMK, resulted in a graduated loss of monkey virulence. Rhesus monkeys inoculated with the PDK 50 strain failed to develop detectable viremia and only 1 of 4 developed an antibody response. Also, replication of PDK 50 was completely shut-off at 39 degrees C. The graduated change in biological properties noted, particularly those in PDK cells, provide a range of potential vaccine candidates for evaluation in human beings.
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