The successful use of the baculovirus system depends upon the production of a high quality virus stock which is accurately titrated: both are readily achieved. Baculovirus stocks should be prepared by infection of Sf-9 (or other cell) cultures with recombinant virus at a low multiplicity of infection (MOI, 0.01-0.05 infectious particles per cell) in order to minimize production of defective interfering particles (DIPs). Infection at high MOI leads to increased levels of defective particles and a deterioration in performance of the virus stock (Kool et al. 1991; Wickham et al. 1991). The cell population will increase by about two-fold until the initial baculovirus replication has produced sufficient progeny virus to infect all the cells (approx 24 h p.i.), and cell division ceases. After infection, cell metabolism increases, and the nutrient requirements per cell will be approximately double that of non-infected cells. It is therefore important that cultures be maintained throughout at a maximum of about half the cell concentration achievable during cell growth (<1.5 X 106 cells/ml). Baculovirus stocks should be harvested 4, or at the latest 5, days p.i. in order to minimize the accumulation of viral proteases and other degradative enzymes. Cells and debris should be removed by centrifugation (approx. 1000 g for 5 min), and the supernatant stored in the dark at 4°C. Foetal bovine serum (FBS) at 2-5 % is normally added to improve stability, and stocks containing FBS can be stored for years without significant reduction in titre. If serumfree virus stock is required, infected cells may be resuspended in serum-free medium once cell multiplication has ceased (1 to 2 days p.i.), the supernatant harvested at day 4 p.i. and stored in the dark at 4 °C. Such preparations should not deteriorate significantly over a short period of time (1-2 months), but should be reassayed immediately before use, to determine titre.
The traditional method for estimation of infectious baculovirus concentrations is the plaque assay: monolayers of susceptible cells are infected with dilutions of the virus, and the number of infectious foci determined visually by counting plaques (areas of dead cells) after 5 or 6 days (Griffiths & Page 1977). The assay requires considerable skill to achieve accurate results, and adds a significant delay to the overall production process. A modified assay, in which early foci are detected by an antibody against the virus protein gp64, allows titres to be estimated after 2-3 days (Kitts & Green 1999). More recently, a technically simple assay based on the inhibition of growth
MOI (infectious virus particles per cell) Figure 7.1 Effect of MOI on the proportion of insect cells infected by baculovirus.
of insect cells has been shown to give very rapid (18 h) and accurate results (see Appendix). Since viral infection rapidly (2-3 h p.i.) halts cell division, the degree of growth inhibition compared with controls readily allows estimation of the number of infectious particles added.
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