Genetic Enhancement of Baculovirus Stability in Continuous CultureEPA Grant Number: R831458
Title: Genetic Enhancement of Baculovirus Stability in Continuous Culture
Investigators: Bonning, Bryony C. , Feiss, Michael G. , Murhammer, David W.
Institution: Iowa State University , University of Iowa
EPA Project Officer: Richards, April
Project Period: January 1, 2004 through December 31, 2006 (Extended to December 31, 2007)
Project Amount: $150,000
RFA: Technology for a Sustainable Environment (2003) RFA Text | Recipients Lists
Research Category: Sustainability , Pollution Prevention/Sustainable Development
Environmentally benign alternatives to chemical insecticides are required to reduce the deleterious impact of these agents on nontarget organisms. Insect viruses, i.e. baculoviruses, are one of the most promising biological agents since they are relatively host specific and do not adversely affect the environment. The long-term goal of the proposed collaborative research is to develop a more cost-effective method for mass-production of baculovirus insecticides. This method involves continuous production of baculoviruses in cell culture without production of few polyhedra (FP) mutant accumulation. FP mutant accumulation results from mutation of the baculovirus fp25k gene that results in loss of FP25K protein expression. Transposon insertion at specific sites in fp25k results in the FP genotype. The hypothesis to be tested in the Bonning lab is that FP mutant accumulation in cell culture can be overcome by modification of the baculovirus gene fp25k. The goal of this research is to generate a genetically stable virus to prevent the negative impact of serial passage on the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV).
Based on the fact that transposon insertion into the fp25k gene occurs at TTAA target sites, we plan to remove the TTAA sites by site-directed mutagenesis. We will test the hypothesis that disruption of fp25k by transposon insertion will be prevented by mutation of the TTAA target sites. Following production of the modified baculovirus and preliminary characterization in the Bonning lab, the viruses will be amplified and tested for stability on repeated passaging in vitro and in the continuous bioreactor system in theMurhammer lab.
We expect 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. All of the modifications that cause FP production in AcMNPV can be explained by transposon activity. Acquisition of a virus that is stable in a continuous bioreactor system will be a key step toward use of this cost-effective method for large-scale production of baculovirus insecticides. Increased use of baculovirus insecticides in integrated pest management will help minimize economic losses resulting from insect pests, without the negative environmental impact associated with classical chemical insecticides.
Successful completion of this project will pave the way for cost-effective large scale in vitro production of baculovirus insecticides. This method will increase the likelihood that baculovirus insecticides will be marketed and used more widely for management of agricultural insect pests, thereby reducing the amount of chemical insecticides required for pest management.