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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

Scott B. Halstead^*, Nyven J. Marchette^*, Arwin R. Diwan^*, Nicholas E. Palumbo, Ravithat Putvatana^* AND Linda Kay Larsen^*

^* Department of Tropical Medicine and Medical Microbiology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96816
Department of Comparative Medicine, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822

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°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.

Accepted for publication January 6, 1984.

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