Development of dengue virus plaques under serum-free overlay medium.
An improved plaque assay for dengue virus was developed utilizing baby hamster kidney (BHK-21) cells initially grown in shaker culture. Different media preparations were tested for uniform and fast formation of BHK-21 cell sheets. Several overlay formulas were tested to develop a rapid plaque assay in 6- and 24-well plastic plates. The best results were obtained utilizing Eagle minimal essential medium (pH 7.2 to 7.4) supplemented with 1 mg of NaHCO3 per ml and 5% newborn calf serum for the formation of cell monolayers after 8 to 24 h of incubation at 37 degrees C. Serum-free Eagle minimal essential medium supplemented with 1% methylcellulose and buffered with 10 mM N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (pH 7.4 to 7.6) was used as an overlay medium. This system allowed for plaque formation after 3 days of incubation of dengue type 2 virus and after 4 days for dengue type 1 and 4 viruses.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003470:Culture Media; D003716:Dengue Virus / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D008274:Magnesium / Q000494:pharmacology; D010948:Viral Plaque Assay / Q000379:methods*
D003470:Culture Media; D008274:Magnesium