Dengue-Virus Recovery by Direct and Delayed Plaques in LLC-MK2 Cells

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Abstract
Summary A method for detecting and propagating dengue viruses in LLC-MK2 cells has been developed by direct plaquing and by fluid maintenance of inoculated cultures 7 to 14 days before plaquing (delayed plaquing). This procedure was used in support of studies of hemorrhagic fever and has resulted in the recovery of 69 dengue viruses, including all four serotypes. About half of these were detected by delayed plaques when direct plaques were not seen. Most of these dengue strains were inoculated simultaneously into mice and cell cultures. The direct and delayed plaque method was more sensitive and efficient than the use of suckling mice. Comparisons of the direct and delayed plaque method with other methods of recovery of dengue virus are discussed. Early in the study, occasional difficulty in preparing low-passage cell-culture seed virus of adequate titer was encountered. Consequently, the growth of dengue virus in LLC-MK2 cells was studied. Virus growth was synchronous and cyclic. In order to avoid inadvertent harvesting of virus during eclipse, live cells, rather than cell lysates, were passaged. This has resulted in consistent production of low-passage seed-virus suspensions.