Grantee Research Project Results

2007 Progress Report: 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 Period Covered by this Report: January 1, 2007 through December 31, 2008
Project Amount: $320,000
RFA: Technology for a Sustainable Environment (2003) RFA Text | Recipients Lists
Research Category: Pollution Prevention/Sustainable Development , Sustainable and Healthy Communities

Objective:

The long-term goal of this research is to develop a more cost-effective method for mass producing baculovirus insecticides. This involves developing methods to overcome the accumulation of few polyhedra (FP) mutants that would otherwise occur upon repeated baculovirus passage in cell culture. This includes two approaches. The first approach is to modify the base sequence of the FP25K gene (a mutation of which leads to FP mutants). This research is being conducted in the Bonning laboratory (Iowa State University). The second approach is to express the FP25K protein from the host cell genome. This research is being conducted in the Murhammer laboratory (University of Iowa).

Progress Summary:

Clone Screening

We have developed a new sensitive and high-throughput technique, combining a 96-well plate end point dilution assay and an immunofluorescent assay, for screening the clones expressing FP25K protein, as detailed below.

Growing the clonal cell line in a 96-well plate in presence of selective reagent (blasticidin).
Infecting the clones on glass cover slips in a 24-well plate with AcMNPV (FP25K deleted).
Staining with primary antibody against FP25K and fluorescent secondary antibody.
Selection of clones using confocal microscopy.

Passaging Experiment with the Stabilized Virus

We have performed the “passaging experiment” using a stabilized virus obtained from the Bonning laboratory (Iowa State University). In particular, the fp25k gene of WT AcMNPV was modified by removing the TTAA sites using template-directed ligation and PCR. In our experiment, the three types of viruses (WT, stabilized, and delta (fp25k gene deleted) AcMNPV) were passaged through the 25th passage in Sf-21 cells in 120-mL shaker flasks. The details of sample analysis are as follows.

Immunofluorescent assay and Western blot: With an increasing number of passages, the number of cells having FP25K protein was observed to be much higher in the case of stabilized AcMNPV than in the case of WT AcMNPV.
PCR analysis: The fp25k gene region shows that the size of the fp25k gene is the same at the 1st, 6th, 11th, 15th and 25th passages for both the WT and stabilized virus. Further analysis will be carried out by DNA sequencing the fp25k gene to check for point mutations.
Polyhedra counting using hemacytometer: Polyhedra production (biopesticide) is seen to be more for the stabilized virus than the WT virus up to the 16th passage.

Immunostaining Assay for Measuring Baculovirus Infectivity

A simple and fast infectivity/titration assay has been developed in a six-well plate using the concept of immunostaining (using the antibody for the gp64 surface protein of baculovirus) to measure the infectivity of the baculovirus particles as follows.

Infecting the insect cells in a on a six-well plate with baculovirus.
Staining with primary antibody against gp64 and enzyme-tagged secondary antibody.
Color development by adding the substrate.
Counting the colored plaques.

Future Activities:

Stabilized viruses (with no TTAA transposon sites) and recombinant insect cells (expressing FP25K protein) will be passaged in a shaker flask and continuous bioreactor system by examining the characteristics of FP and defective interfering particle (DIP) mutations and polyhedra productivity. Specifically, the following will be monitored.

Cell growth and cell viability using a Vi-Cell (Beckman Coulter).
Quantification of virus: Infectivity of the baculovirus using end point dilution assay/immunostaining. using the gp64 antibody/statistical analysis of cell diameter/RT-PCR.
Polyhedra production characterization: Characterization of polyhedra by Transmission Electron Microscopy (TEM) and counting them by hemacytometer.
Identification of FP mutation (proteomic analysis): By Western blot/Immunofluoroscence assay (Confocal microscopy)
Genetic analysis of FP mutation: PCR analysis and DNA sequencing of plaque purified FP mutants from WT and stabilized virus
Identification of DIP mutation using TEM/pulse field gel electrophoresis

Journal Articles:

No journal articles submitted with this report: View all 10 publications for this project

Supplemental Keywords:

biopesticide, continuous production, baculovirus,, Sustainable Industry/Business, RFA, Scientific Discipline, TREATMENT/CONTROL, Technology for Sustainable Environment, Sustainable Environment, Environmental Chemistry, Biochemistry, Technology, baculovirus, biotechnology, electron microscopy, bioinsecticides, bioengineering, agriculture

Progress and Final Reports:

Original Abstract

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.