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Final 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 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 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 and subsequent loss of FP25K protein expression. Transposon insertion at specific sites in fp25k results in the FP genotype. The hypothesis 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 and FP mutant accumulation on the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV).

Summary/Accomplishments (Outputs/Outcomes):

We constructed a recombinant baculovirus that expresses a stabilized version of fp25k in place of the native fp25k. We removed the 13 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 is prevented by mutation of the TTAA target sites. (See R831421 for results). The results showed a reduced incidence of the FP phenotype in late passages of the stabilized virus when compared with the wild type virus at passage 15, but could not eliminate the FP phenotype in very late passages (passage 30).

We received virus samples from passage 0 and passage 25 of AcMNPV clone E2 (Wt0 and Wt25 respectively) and the FP25K stabilized virus (Stab0 and Stab25 respectively) from the University of Iowa and produced occlusion bodies for bioassay against the tobacco budworm, Heliothis virescens to address whether modification of the fp25k gene affected 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. Lethal concentration bioassays were conducted using the droplet feeding method with neonate larvae, with three replicates of 30 larvae per virus dose (five doses). LC50s were calculated using POLO probit analysis and compared by standard lethal dose ratio comparison. The LC50 values indicated that the virulence of both viruses decreased significantly after 25 passages in cell culture. There was no significant difference between the LC50 values of Wt25 and Stab25. This result indicates that modification of the fp25k gene to remove transposon insertion sites did not prevent the reduction in virulence associated with repeated passaging in cell culture.

Conclusions:

The fp25k gene that is susceptible to transposon insertion resulting in the FP baculovirus phenotype was modified to remove the 13 potential transposon insertion sites. This stabilized gene was inserted into the genome of AcMNPV E2 and tested for stabilization on repeated passaging in cell culture. While the stabilized gene reduced the incidence of the FP phenotype at passage 15 when compared to the wild type virus, it did not eliminate the FP phenotype at passage 30. Bioassays of virus derived from passage 0 and passage 25 showed that the virulence of both wild type AcMNPV E2 and the stabilized virus had decreased significantly by passage 25. Elimination of the transposon insertion sites from the fp25k gene did not stabilize the virus, indicating that virulence was impacted by other destabilizing mechanisms that occurred on repeated passaging in cell culture.

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
2006 Progress Report
2007 Progress Report
Original Abstract

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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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