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

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 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 smallplaque 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 attenuation characteristics of the S-1 small-plaque subline make it appear suitable as a vaccine candidate virus. 1221 on August 1, 2020 by guest

Sequence of the dengue virus type 2 (strain PR-159) NS1 gene and comparison with its vaccine derivative

“…Although the basis of attenuation is unknown, by comparing genome sequences of these isolates and their culture derivatives, it may be possible to determine molecular markers of virulence. The dengue virus type 2 (DEN-2), strain PR159, was isolated in Puerto Rico (1969) from a patient with dengue fever and subsequently passaged 19 times in cell culture (12,13). This PR-159-S1 strain is a small-plaque, temperature-sensitive derivative, which exhibits reduced virulence for suckling mice and is markedly attenuated in rhesus monkeys.…”

Lack of greater seroconversion of rhesus monkeys after subcutaneous inoculation of dengue type 2 live-virus vaccine combined with infection-enhancing antibodies

“…Dengue virus serotypes 1, 2, 3, and 4 have long been associated with epidemics of dengue fever in tropical areas of the world. Widespread epidemics of dengue hemorrhagic fever have been observed in Southeast Asia (18).…”

“…Widespread epidemics of dengue hemorrhagic fever have been observed in Southeast Asia (18). Because dengue fever constitutes a major health problem in these geographical regions, several laboratories have attempted to develop live, attenuated vaccines effective against this viral disease (4,12,17,18). A clone of dengue virus serotype 2 is presently being used for the preparation of such a vaccine, which has been designated as DEN-2/S-1 (4,5).…”

“…Because dengue fever constitutes a major health problem in these geographical regions, several laboratories have attempted to develop live, attenuated vaccines effective against this viral disease (4,12,17,18). A clone of dengue virus serotype 2 is presently being used for the preparation of such a vaccine, which has been designated as DEN-2/S-1 (4,5). The vaccine virus has reduced animal virulence (11), is temperature sensitive (4), and shows no evidence of reversion to the wild-type characteristics of the dengue type 2 virus (4, 5, 11).…”

“…A clone of dengue virus serotype 2 is presently being used for the preparation of such a vaccine, which has been designated as DEN-2/S-1 (4,5). The vaccine virus has reduced animal virulence (11), is temperature sensitive (4), and shows no evidence of reversion to the wild-type characteristics of the dengue type 2 virus (4, 5, 11).…”