Utilizing the virus-induced blocking of apoptosis in an easy baculovirus titration method

Abstract

Baculovirus-mediated protein expression is a robust experimental technique for producing recombinant higher-eukaryotic proteins because it combines high yields with considerable post-translational modification capabilities. In this expression system, the determination of the titer of recombinant baculovirus stocks is important to achieve the correct multiplicity of infection for effective amplification of the virus and high expression of the target protein. To overcome the drawbacks of existing titration methods (e.g., plaque assay, real-time PCR), we present a simple and reliable assay that uses the ability of baculoviruses to block apoptosis in their host cells to accurately titrate virus samples. Briefly, after incubation with serial dilutions of baculovirus samples, Sf9 cells were UV irradiated and, after apoptosis induction, they were viewed via microscopy; the presence of cluster(s) of infected cells as islets indicated blocked apoptosis. Subsequently, baculovirus titers were calculated through the determination of the 50% endpoint dilution. The method is simple, inexpensive, and does not require unique laboratory equipment, consumables or expertise; moreover, it is versatile enough to be adapted for the titration of every virus species that can block apoptosis in any culturable host cells which undergo apoptosis under specific conditions.

Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin

“…5 Baculovirus titration is necessary to determine the optimal dose for infecting host cells. 5 Researchers have reported various assays for the quantifying viruses, including plaque assay, 5 end-point dilution assay, 6,7,8 and 50% tissue culture infectivity dose assay. 9 However, current baculovirus titration methods are major timeconsuming processes, requiring 6-10 days for plaque formation, 10 which increases the risk of cell damage.…”

Image-Based Cell-Size Estimation for Baculovirus Quantification

“…Although these assays are useful, each has its drawbacks (17). A comparison of different methods revealed that the cell-size assay (18) performs well with a reasonable standard error (SE) and is recommended for the determination of virus titer (16).…”