Optimization of an assay for baculovirus titer and design of regimens for the synchronous infection of insect cells

We have previously described a quantitative model of the trafficking of baculovirus in insect cells that considers the various infection steps 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 inefficiency of existing assays for virus titer, and to develop a modified end-point dilution assay optimized to measure more completely the concentration of intrinsically infectious particles in a virus inoculum. The titer obtained from existing assays incompletely counts infectious virus particles due primarily to the incomplete adsorption of the virus during the short, standard 1-h incubation period. For representative assays, the calculated bound virus is generally about 10% of the added virus, but could be as low as 1.4%, underestimating actual titers by 3-70-fold. A modified end-point dilution assay involving centrifugation has been developed from both quantitative and qualitative analyses. The ratio of particle to plaque-forming unit with the optimized assay was 4-6 compared to 100-300 for typical assays, representing a significant improvement in the measurement of total infectious virus concentration.