Grantee Research Project Results
2007 Progress Report: Genetic Enhancement of Baculovirus Stability in Continuous Culture
EPA Grant Number: R831458Title: 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 Period Covered by this Report: January 1, 2007 through December 31, 2008
Project Amount: $150,000
RFA: Technology for a Sustainable Environment (2003) RFA Text | Recipients Lists
Research Category: Sustainable and Healthy Communities , Pollution Prevention/Sustainable Development
Objective:
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 among the most promising biological agents because they are relatively host-specific and do not adversely affect the environment. The long-term goal of the proposed 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 laboratory 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).
Progress Summary:
Construction of Recombinant Virus Expressing Stabilized fp25k
We constructed a recombinant baculovirus that expresses a stabilized version of fp25k in place of the native fp25k. We removed the TTAA sites, which serve as transposon target sites from the gene. This stabilized virus was tested alongside the wild type virus AcMNPV clone E2 for stability on passaging in cell culture at the University of Iowa to test the hypothesis that disruption of fp25k by transposon insertion will be prevented by mutation of the TTAA target sites. (See U.S. Environmental Protection Agency Grant Number R831421 for results.)
We received virus samples from passage 0 and passage 25 of AcMNPV clone E2 and the FP25K stabilized virus from the University of Iowa and amplified these viruses in cell culture for production of sufficient viral occlusion bodies for insect bioassay. We are currently testing the insecticidal efficacy of the stabilized virus in the tobacco budworm, Heliothis virescens, to address whether modification of the fp25k gene affects in vivo activity of the virus (LD50). Because the long-term goal of this project is to produce virus for insect pest control purposes, maintenance of insecticidal efficacy of the virus is of the utmost importance.
Conclusions
The fp25k gene that is susceptible to transposon insertion resulting in the FP baculovirus phenotype has been modified to remove the 13 potential transposon insertion sites. This stabilized gene has been inserted into the genome of AcMNPV and has been tested for stabilization on repeated passaging in cell culture. Determination of the potential impact of fp25k modification on the insecticidal efficacy of the virus is underway.
Journal Articles:
No journal articles submitted with this report: View all 3 publications for this projectSupplemental Keywords:
pathogens, pollution prevention, clean technology, genetics, agriculture,, RFA, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Sustainable Environment, Environmental Chemistry, Genetics, Technology, Technology for Sustainable Environment, clean technologies, cleaner production, environmental sustainability, alternative materials, insecticide production, agriculture, environmentally benign alternative, baculovirusRelevant Websites:
http://www.ent.iastate.edu/dept/faculty/bonningb/ Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.