Plaque assay and improved yield of human coronaviruses in a human rhabdomyosarcoma cell line

Abstract

Propagation and plaque assay of human coronavirus prototypes were studied in two human cell lines: a diploid fetal tonsil (FT) and a heteroploid rhabdomyosarcoma (RD) cell lines. Plaques, observed within 2 to 3 days on FT cell monolayers with both 229E and OC43 viruses, appeared as colorless areas after staining with neutral red or crystal violet, whereas neutral red staining was required for visualization of plaques on RD cells. The plating efficiencies were approximately equal between the two cell lines, but virus assay by plaque formation was 15- to 30-fold more efficient than tube dilution assay with 50% endpoints. The discrepancy between 50% endpoint and plaque-forming unit values was striking and appeared to result from the fact that killing of cells (particularly RD cells) by coronaviruses was not accompanied by visible changes in the cells but killing was detected by the failure of infected cells to stain with a vital dye. The latent phase in one-step growth curves was 5 to 6 h for both viruses in either cell line, but the maximum yield of intracellular virus was reached in 18 to 20 h for FT cells and 24 to 28 h for RD cells. Virus release also differed between the two cell lines: in FT cells, the maximum yield of extracellular virus was reached 2 to 3 h later than that of intracellular virus, whereas in RD cells, the difference was 5 h for 229E virus and 10 h for OC43 virus. Although both cell lines appear equally useful for plaque assay, RD cells would be preferred for mass virus propagation because yields (5 X 10(8) plaque-forming units per ml) were 10-fold higher than in FT cells, a finding true for both virus prototypes.