Grantee Research Project Results
Final Report: Collaborative Research: Cost-Effective Production of Baculovirus Insecticides (TSE03-D)
EPA Grant Number: R831421Title: 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 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:
Chemical pesticide use has many undesirable consequences, including the development of insecticide resistance, reduction of soil fertility, and adverse health effects on non-target organisms. Therefore, it is highly desirable to reduce chemical pesticide use through utilization of integrated pest management (IPM) strategies. An important element of an IPM strategy is the use of biocontrol agents, including baculoviruses. Baculoviruses have many desirable characteristics, including the fact that they do not kill beneficial insects and are environmentally benign. However, low cost, large-scale baculovirus production using continuous insect cell culture is seriously hindered by undesirable mutations in the baculovirus genome. Few polyhedra (FP) and defective interfering particle (DIP) mutants are commonly responsible for the reduction in occluded virus yield and decreased infectivity, which is unacceptable for bio-insecticides. The FP25K protein is generally missing from the FP mutants, which commonly results from the insertion of transposons (DNA fragments from the host insect cell genome) into the baculovirus fp25k gene. The current research used Autographa californica multiple nucleopolyhedrovirus (AcMNPV) to test the hypothesis that FP mutant accumulation can be reduced by removing the transposon target sites from the fp25k gene of the wild type AcMNPV genome. It was demonstrated that removal of the transposon target sites from the wild type baculovirus fp25k gene (named as Acfp25km(TTAA)) stabilized the FP25K protein expression and delayed the incidence of the FP phenotype from passage 5 to passage 10. Electron micrographs of infected cells revealed that more virus particles were found inside the nucleus in the Acfp25km(TTAA) compared to WT AcMNPV, but abnormalities were observed in nucleocapsid envelopment and virus particle occlusion in polyhedra. A significant loss in FP25K protein expression was observed in cells infected with WT AcMNPV by passage 12, whereas cells infected with Acfp25km(TTAA) had stable FP25K protein expression through 32 passages (the highest passage investigated in this research). Hence, removing the transposon insertion sites in the fp25k gene (i.e., Acfp25km(TTAA)) resulted in stable FP25K protein expression, but mutations elsewhere in the baculovirus genome still lead to FP mutant accumulation, although this accumulation was delayed compared to the case with the wild type baculovirus. Furthermore, a simple and novel assay based on restriction enzyme digestion of baculovirus DNA and pulse field gel electrophoresis was developed to detect and quantify DIP mutants. This methodology was used to demonstrate that DIP mutant accumulation was delayed for Acfp25km(TTAA) compared to WT AcMNPV, thereby suggesting a relationship between FP and DIP mutations.
Background. Bio-pesticides are useful in overcoming the significant disadvantages of chemical pesticides, such as detrimental effects on non-target organisms, higher animals, and soil fertility. However, the current production cost of potential biopesticides, such as the baculovirus (which kills only targeted insects and is environmentally benign), is significantly higher than that of chemical pesticides. Costs could be significantly reduced by using a continuous production process. Unfortunately, traditional continuous processes are not feasible due to accumulation of few polyhedra (FP) and defective interfering particle (DIP) mutants that render the product useless as a biopesticide. In this context, our primary goal was to overcome the FP mutation, resulting, generally, from the insertion of host cell DNA sequences, known as transposons, into the baculovirus fp25k gene. In addition, we examined whether FP mutants are a necessary precursor of the DIP mutants.
Objectives. The long-term goal of this research was to develop a more cost-effective method for mass producing baculovirus insecticides. The specific objective of the research described in this project involved developing methods to overcome the accumulation of few polyhedra (FP) mutants that would otherwise occur upon repeated baculovirus passage in cell culture. This included two approaches. First, the base sequence of the fp25k gene (whose mutation leads to FP mutants) in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was modified to remove all 13 of the TTAA transposon insertion sites in the fp25k gene. This was done in a manner that maintained the same amino acid sequence and thus produced the same FP25K protein. The ability of the resulting baculovirus (named Acfp25km(TTAA)) to resist FP and DIP mutant accumulation upon passaging in cell culture was investigated in detail. Second, expressing the FP25K protein from the host cell genome.
Summary/Accomplishments (Outputs/Outcomes):
Research Results
Effect of Acfp25km(TTAA) on Few Polyhedra (FP) Mutant Accumulation. Characteristics of the FP phenotype include the presence of few (less than 10) polyhedra per baculovirus infected insect cell and the presence of few to no virus particles per polyhedra. First, it was demonstrated that there was no statistically significant differences of polyhedra production, FP25K protein expression, and toxicity to insects between WT AcMNPV and Acfp25km(TTAA) at passage 1. Second, there was a significant difference between Acfp25km(TTAA) and WT AcMNPV in regards to the percentage of cells having polyhedra after 5 passages. By passage 15, however, there was no significant difference between these two baculoviruses, i.e., both of them had a high percentage of cells lacking polyhedra. Similarly, most of the WT AcMNPV polyhedra at passage 5 contained few to no virus particles, while most of the Acfp25km(TTAA) polyhedra contained many virus particles. By passage 10, however, both Acfp25km(TTAA) and WT AcMNPV polyhedra were largely void of virus particles. Third, it was demonstrated that FP25K protein expression was decreased by passage 12 in cells infected with WT AcMNPV, but remained stable in cells infected with Acfp25km(TTAA) until passage 32 (the latest passage investigated). These studies utilized western blots and a unique immunofluorescence confocal microscopy assay in which cells were co-stained to detect baculovirus infection (through expression of the gp64 protein on the cell surface) and FP25K protein expression. This latter assay provided the means to determine, e.g., the percentage of infected cells that expressed FP25K. Fourth, the effectiveness (measured as LC50) of the WT AcMNPV and Acfp25km(TTAA) polyhedra as biopesticides were both found to decline significantly from the first to 25th passage. Furthermore, there was no significant difference in the effectiveness of the WT AcMNPV and Acfp25km(TTAA) polyhedra at passage 25. Unfortunately, the effectiveness of the polyhedra was not investigated at intermediate passages where it would be expected that Acfp25km(TTAA) polyhedra would be superior to WT AcMNPV polyhedra.
Effect of Acfp25km(TTAA) Defective Interfering Particle (DIP) Mutant Accumulation. Baculovirus DIP mutants generally lack ~43% of their genome and some of the genes in the deleted genome are essential for baculovirus replication. Therefore, DIP mutants will only replicate in cells that are co-infected by a “normal” baculovirus that contains these essential genes. First, a modified restriction enzyme analysis method was developed that utilized pulse field gel electrophoresis. This approach provides the means to qualitatively and quantitatively characterize DIPs. Prior to electrophoresis, the viral DNA was linearized by digestion with AvrII that cuts the circular AcMNPV genome at single site. Prior to the development of this method DIP analysis was limited to restriction enzyme analysis or electron microscopic visualization. Both of these methods are extremely time consuming and labor-intensive, and provide only limited information. Second, this method was used to evaluate DIP formation resulting from passaging WT AcMNPV and Acfp25km(TTAA) in insect cell culture. Specifically, it was demonstrated that (i) at passage one both WT AcMNPV and Acfp25km(TTAA) were mostly of standard size DNA (134 kbp) and (ii) by passage 16 three distinct bands (110, 97, 82 kbp) were observed for WT AcMNPV with 50% of total DNA being from DIPs, but only 20% of the Acfp25km(TTAA) DNA was from DIPs; two bands (110, 90 kbp) were observed. Third, these and other results clearly demonstrated that DIP formation was delayed (compared to WT AcMNPV) with Acfp25km(TTAA) and that a different mechanism was involved. For example, by passage 5 WT AcMNPV showed the presence of 82 kb DIPs (i.e., d43 - DIPs with 43% deletion), while no detectable levels of d43 were present with Acfp25km(TTAA). Furthermore, from passages 5 through 16 the amount of d43 was significantly higher for WT AcMNPV than Acfp25km(TTAA). Fourth, electron microscopy analysis demonstrated an heterogeneous budded virus population. The mean size and standard deviation (n = 89) of WT AcMNPV at passages 1 and 30 were 288 ± 26 and 196 ± 44 nm, respectively. At passage one 99% of the virus population was > 200 nm, while only 38% of the virus was this large by passage 30. Furthermore, the budded virus had a significantly wider size range by passage 30.
FP25K Protein Expression From the Insect Cell Genome. The general idea behind this approach was to insert the fp25k gene into the insect cell genome. Thus, the fp25k gene would not mutate as does the corresponding gene in the AcMNPV genome. This belief was based primarily on the lack of selection pressure of any mutation that might occur in the cell-based version. The keys to making this approach work include (i) having a promoter that is turned on at the proper time post-infection and (ii) having an appropriate FP25K protein expression level. PCR was used to demonstrate that the fp25k gene under control of the p6.9 promoter was successfully inserted into the insect cell genome. This promoter was chosen since it is turned on at essentially the same time post-infection as the native fp25k gene promoter in AcMNPV and is of comparable strength. Unfortunately, none of the isolated clones expressed detectable levels of FP25K protein when infected with an AcMNPV lacking the fp25k gene. A possible reason for the lack of FP25K protein expression is that the baculovirus RNA polymerase (which is involved with the expression of genes under control of late baculovirus promoters like p6.9) cannot access the fp25k gene within the cell genome. It is important to note, however, that experiments were not conducted to detect the fp25k mRNA.
Relevance and Future Work
The results of this research demonstrate that FP25K protein expression can be stabilized by removing the TTAA transposon insertion sites from the fp25k gene. In spite of this, however, FP and DIP mutant accumulation, while delayed, still occurs upon passaging in insect cell culture. Therefore, the original goal of developing a method to mass produce baculovirus in a continuous system has not yet been achieved. This research in combination with previous work by other investigators has, however, given strong evidence that mutations in other genes are involved in FP mutant accumulation. Note that there is compelling evidence from previous work that DIP mutant accumulation can be eliminated through bioreactor design that takes advantage of the fact DIPs can only replicate in cells that are also infected with a “normal” baculovirus. That is, initially infecting cells at a multiplicity of infection (MOI) low enough to avoid co-infection of cells with a DIP and normal baculovirus will eliminate DIP replication. Therefore, future research must initially focus on eliminating FP mutant accumulation and then research can focus on developing a suitable bioreactor design. To this end, future research should involve evaluating the Acfp25km(TTAA) FP mutants to determine which genes are mutated. Based on previous research likely candidates include the da26, 94k, and polyhedrin genes. After the mutated genes are identified, then approaches would be taken to stabilize them. The specific approach taken would be dependent upon the nature of the mutations.
Human Resource Development
Two students at the University of Iowa working on this project received significant training in insect cell culture, baculovirus procedures, molecular biology, etc. Specifically, Lopamudra Giri, a graduate student, and Aaron Irons, an undergraduate students worked on this project. Dr. Giri received a Ph.D. degree in May 2009 and has started a postdoctoral position at the University of Illinois, Urbana-Champaign. Aaron Irons will be a senior at the University of Iowa during the 2009-10 academic year and is on schedule to graduate with a B.S. degree in May 2010. His future plans include attending graduate school to pursue a Ph.D.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 10 publications | 2 publications in selected types | All 2 journal articles |
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Type | Citation | ||
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Giri L, Li H, Sandgren D, Feiss M, Roller R, Bonning BC, Murhammer DW. Removal of transposon target sites from the Autographa californica multiple nucleopolyhedrovirus fp25k gene delays, but does not prevent, accumulation of the few polyhedra phenotype. Journal of General Virology 2010;91(12):3053-3064. |
R831421 (Final) |
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Giri L, Feiss MG, Bonning BC, Murhammer DW. Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k. Journal of General Virology 2012;93(2):389-399. |
R831421 (Final) |
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Supplemental Keywords:
biopesticide, continuous production, baculovirus, RFA, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Environmental Chemistry, Sustainable Environment, Technology, Technology for Sustainable Environment, Biochemistry, bioengineering, biotechnology, insecticide production, agriculture, baculovirus, bioinsecticidesProgress 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.
Project Research Results
- 2007 Progress Report
- 2006 Progress Report
- 2005 Progress Report
- 2004 Progress Report
- Original Abstract
2 journal articles for this project