Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. III. Reversion to virulence by passage of cloned virus in fetal rhesus lung cells

Abstract

Two strains of primary dog kidney-passaged dengue (DEN) 4 (H-241) virus cloned by terminal dilution (PDK 24-TD3 and 35-TD3) were propagated in fetal rhesus lung (FRhL) cells to produce candidate vaccine virus seeds. Both serial passage and prolonged replication of PDK 24-TD3 in FRhL resulted in appearance of medium and large plaques in LLC-MK2 assays. When picked, these plaques proved to contain temperature-resistant, monkey-virulent revertants. Serial passage and prolonged replication of PDK 24-TD3 in LLC-MK2 cells did not result in reversion; but, prolonged replication in PDK cells did. Passage of PDK 35-TD3 in FRhL cells resulted in appearance of medium size plaques which, when picked, yielded temperature sensitive (ts) (38.5 degrees C) viruses of low monkey-virulence. Because of its stability in monkeys and FRhL cells, reduced monkey virulence and ts property. PDK 35-TD3 is a promising candidate for trial in man.