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

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Abstract
Dengue virus, type 2, in viremic human sera and after passage in cell cultures produces mixtures of small and large plaques when assayed in [rhesus monkey kidney] 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 had lower nonpermissive temperatures than the parent virus by 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 and temperature-sensitive and attenuation characteristics of the S-1 small-plaque subline make it appear suitable as a vaccine candidate virus.