Dengue virus plaque development in simian cell systems. II. Agar variables and effect of chemical additives

Dengue type 2 virus, strain New Guinea B, plaqued with equal facility and titer under overlays containing six different grades of commercial agar in the LLC-MK(2) cell system. Doubling the agar volume on LLC-MK(2) cell monolayers increased the plaque development time of dengue type 1, strain Hawaii. Storage of agar at 56 C reduced or totally abolished dengue type 4, strain H-241, plaque titer in LLC-MK(2) cells. The influence of six known virus plaque-enhancing compounds on plaque development of all dengue virus serotypes was studied in two continuous simian kidney cell lines, LLC-MK(2) and Vero. In the absence of any chemical additive, plaque development of all dengue serotypes was more rapid (4 to 10 days) in the LLC-MK(2) line than in the Vero line (6 to 13 days). Increased plaque development time of type 1, strain Hawaii, by pancreatin and plaque-size doubling of dengue types 1 and 4 was the only advantage conferred by the addition of six chemical additives in the LLC-MK(2) cell system. Dengue types 1 and 6 failed to plaque in the Vero cell system unless aided by a plaque-enhancing compound; plaques of dengue types 2, 3, 4, and 5 appeared sooner (2 days) and were increased in plaque diameter. The optimal DEAE concentration for plaquing dengue type 1, strain Hawaii, was 100 mug/ml; plaque development either failed at lower concentrations or was inhibited at higher (200 mug/ml) concentrations.