Isolation of a temperature-sensitive dengue-2 virus under conditions suitable for vaccine development

Dengue virus, type 2, in viremic human sera and after passage in cell cultures produces mixtures of small and large plaques when assayed in LLC-MK2 cells. Clones of dengue virus type 2 obtained by plaque selection in primary green monkey kidney cell cultures were tested for temperature sensitivity in vitro and for virulence by intracerebral inoculation of suckling mice. Sublines of a small-plaque clone were found to have lower nonpermissive temperatures than the parent virus by both plaque formation and release of infectious virus into the culture media. Small-plaque sublines were significantly less virulent in suckling mice than was the parent virus. Sublines from a large-plaque clone were not temperature sensitive and closely resembled parent virus mixed-plaque morphology. When small-plaque sublines were serially passaged using undiluted inocula, reversion occurred as evidenced by the appearance of large plaques and return of mouse virulence. Small-plaque virus could be maintained through several serial passages without reversion by using low-input inocula. Desirable passage history as well as temperature-sensitive and attentuation characteristics of the S-1 small-plaque subline make it appear suitable as a vaccine candidate virus.