Chemically-induced temperature sensitive mutants of dengue virus type 2

Abstract

Summary Temperature sensitive(ts) mutants of dengue virus type 2 (DEN-2, TH-36 isolate) were induced by replication in primary hamster kidney cells treated with 5-azacytidine. Sevents mutants were obtained from 138 clones isolated by an immunofluorescent cloning technique. Of these 7ts mutants, 5 were sufficiently stable to permit partial characterization. Complementation was detected at very low but statistically significant levels between somets mutants at 40° C. Viral double-stranded RNA production was evaluated in LLC-MK2 cells at 30° and 40° C by micro-quantitative complement fixation. The results of complementation tests and RNA production tests indicated that the 4 of 5 stablets mutants constitute 3 separate complementation groups (2 RNA⁺ and 1 RNA⁻ groups), while a fifthts mutant was RNA⁻ but non-complementable. The data presented here indicate that a genetic system can be developed without employing traditional plaque or cytopathology methods. Further, the 5 DEN-2ts mutants are believed to represent the only set of complementation-positive flavivirus mutants so far isolated.