PG-4 cell plaque assay for xenotropic murine leukemia virus

Xenotropic murine leukemia virus (X-MuLV) is often used in retrovirus elimination studies involving rodent cells. Currently, X-MuLV is measured using a focus-forming assay on mink (MiCl1 S+L-) or cat (PG-4 S+L-) cell lines. An easier and quicker PG-4 cell plaque assay, which retains the statistical reproducibility of the focus-forming assay, was developed and evaluated in this study. The PG-4 plaque assay is more sensitive than the MiCl1 focus assay for titering X-MuLV. The best results were achieved by passaging PG-4 cells at a seeding density of 4x10(6) cells/T185 flask twice a week, inoculating 3x10(5) cells/well on six-well plates and performing the assay at 35 degrees C. The overall variability of the assay was 0.30 log10 titer unit. A linear response to dilution was observed for wells containing 5-70 plaques. The limit of quantitation was 10 PFU/ml. Using six wells per dilution, the 95% confidence limit of the grand mean titer was within +/-0.5 log10.