OPTIMIZATION OF AN ASSAY FOR BACULOVIRUS TITER AND DESIGN OF REGIMENSFOR THE SYNCHRONOUS INFECTION OF INSECT CELLS

We have previously described a quantitative model of the trafficking o f baculovirus in insect cells that considers the various infection ste ps such as attachment, internalization, endosomal fusion, and nuclear accumulation. Concepts from the model were used to design synchronous infection regimens for various cell lines, to analyze the inherent ine fficiency of existing assays for virus titer, and to develop a modifie d end-point dilution assay optimized to measure more completely the co ncentration of intrinsically infectious particles in a virus inoculum. The titer obtained from existing assays incompletely counts infectiou s virus particles due primarily to the incomplete adsorption of the vi rus during the short, standard 1-h incubation period. For representati ve assays, the calculated bound virus is generally about 10% of the ad ded virus, but could be as low as 1.4%, underestimating actual titers by 3-70-fold. A modified end-point dilution assay involving centrifuga tion has been developed from both quantitative and qualitative analyse s. The ratio of particle to plaque-forming unit with the optimized ass ay was 4-6 compared to 100-300 for typical assays, representing a sign ificant improvement in the measurement of total infectious virus conce ntration.