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2006 Progress Report: Genetic Enhancement of Baculovirus Stability in Continuous Culture

EPA 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 Period Covered by this Report: January 1, 2006 through December 31, 2007
Project Amount: $150,000
RFA: Technology for a Sustainable Environment (2003)
Research Category: Pollution Prevention/Sustainable Development

Description:

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 one of 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 project 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 objective of this research project 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. The DNA sequence of the insert was confirmed by sequencing. Clones of the recombinant virus were characterized by restriction enzyme digestion to examine the genomic structure of the virus. This stabilized virus was sent to the University of Iowa for testing alongside the wild type virus AcMNPV clone E2 for stability on passaging in cell culture. During passaging, polyhedrin and budded virus production, FP25K expression, and polyhedron structure will be monitored. Through these experiments, we will test the hypothesis that disruption of fp25k by transposon insertion will be prevented by mutation of the TTAA target sites. In the event that the virus is stable on repeated passage, we will test the insecticidal efficacy of the stabilized virus at Iowa State University to address whether modification of the fp25k gene affected in vivo activity of the virus. 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 is now undergoing testing for stabilization on repeated passaging in cell culture.

Future Activities:

The investigators did not report any future activities.

Journal Articles:

No journal articles submitted with this report: View all 3 publications for this project

Supplemental Keywords:

pathogens, pollution prevention, clean technology, genetics, agriculture, , INTERNATIONAL COOPERATION, TREATMENT/CONTROL, Sustainable Industry/Business, Scientific Discipline, RFA, Technology for Sustainable Environment, Sustainable Environment, Technology, Environmental Chemistry, Ecology and Ecosystems, Genetics, pollution prevention, environmental sustainability, cleaner production, clean technologies, baculovirus, insecticide production, agriculture, alternative materials, application of agricultural chemicals
Relevant Websites:

http://www.ent.iastate.edu/dept/faculty/bonningb/ exit EPA

Progress and Final Reports:
2004 Progress Report
Original Abstract
2007 Progress Report
Final Report

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The 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.

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