Collaborative Research: Cost-Effective Production of Baculovirus Insecticides (TSE03-D)EPA Grant Number: R831421
Title: Collaborative Research: Cost-Effective Production of Baculovirus Insecticides (TSE03-D)
Investigators: Murhammer, David W. , Bonning, Bryony C. , Feiss, Michael G.
Institution: University of Iowa , Iowa State University
EPA Project Officer: Richards, April
Project Period: January 1, 2004 through December 31, 2006 (Extended to December 31, 2008)
Project Amount: $320,000
RFA: Technology for a Sustainable Environment (2003) RFA Text | Recipients Lists
Research Category: Pollution Prevention/Sustainable Development , Sustainability
The hypotheses that will be tested in this research are: (i) FP mutant accumulation can be overcome through expression of the FP25K protein from the host insect cell genome; and (ii) FP mutant accumulation can be overcome through modification of the fp25k gene in the baculovirus genome. Hypotheses (i) and (ii) will be tested through addressing Objectives 1 and 2, respectively: (1) overcome FP mutant accumulation in cell culture through expression of FP25K protein from the host insect cell genome; and (2) overcome FP mutant accumulation in cell culture through modification of the fp25k gene.
Accomplishing Objective 1 will involve co-transfecting host insect cells with a plasmid containing the fp25k gene under control of the p6.9 promoter and a plasmid containing an antibiotic resistance gene. Cells will then be selected that are antibiotic resistant and therefore likely to contain the fp25k gene. The presence of this gene in the host cell genome will be verified using standard methods. FP25K protein expression will then be characterized as a function of time post-infection in cells containing the fp25k gene. Furthermore, the polyhedra produced in these cells when infected by FP AcMNPV will be characterized by light and electron microscopy and in vivo testing. These polyhedra will also be compared to polyhedra produced in cells lacking FP25K protein expression that are infected with either MP or FP AcMNPV. Finally, the stability of these cells will be investigated in regards to long term passaging in cell culture and in a 2-stage bioreactor system.
Accomplishing Objective 2 will involve removing the TTAA sites in the fp25k gene by site-directed mutagenesis. Following production of the modified baculovirus and preliminary characterization, the viruses will be amplified and tested for stability on repeated passaging in vitro. Stability in a continuous bioreactor system will also be investigated.
It is expected that host Sf-9 and Tn-5B1-4 cells will be obtained that contain the fp25k gene under control of the p6.9 promoter, and that these cells will produce normal polyhedra, even when infected with AcMNPV of the FP genotype. Furthermore, it is expected that removal of TTAA target sites from AcMNPV fp25k will stabilize the virus on repeated passaging in cell culture by preventing insertion of transposable elements from the host cells into the fp25k gene.
Estimated Improvement in Risk Management: The continuous production system resulting from the proposed research will provide a method of mass-producing baculoviruses at a reduced cost for use as bioinsecticides. This is likely to lead to the increased use of these bioinsecticides, thereby reducing the detrimental effects caused by the chemical insecticides that would otherwise be used.