2004 Progress Report: Genetic Enhancement of Baculovirus Stability in Continuous CultureEPA 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, 2004 through December 31, 2005
Project Amount: $150,000
RFA: Technology for a Sustainable Environment (2003) RFA Text | Recipients Lists
Research Category: Sustainability , Pollution Prevention/Sustainable Development
The overall goal of this research project is to develop a more cost-effective method for mass-production of baculovirus insecticides, which represent an environmentally benign alternative to chemical insecticides for insect pest control. The specific objective of this research project is to generate a genetically stable virus to prevent the negative impact of serial passage in cell culture on the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). A stabilized virus would allow for use of continuous bioreactors for cost-effective production of these 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, which causes 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 fp25k gene, susceptible to transposon insertion resulting in the FP baculovirus phenotype, has been modified to remove the 13 potential transposon insertion sites. This stabilized gene will be inserted into the genome of AcMNPV. A bioassay procedure involving repeated passaging of virus in Sf21 cells has been developed for testing the stability of the modified virus.
Construction of a Stabilized fp25k Gene
Based on the fact that transposon insertion into the fp25k gene occurs at TTAA target sites, we have removed the TTAA sites from the gene. We originally proposed the use of site-directed mutagenesis (Clontech Site-directed mutagenesis kit), but experienced difficulties with this approach. The modified gene was produced by using template-directed ligation and PCR. We constructed full-length versions of the gene. Sequencing of the PCR product indicated the introduction of several errors into the sequence. We used PCR to correct these errors, and we now have the completed, full-length sequence of the modified fp25k.
Establishment of a Bioassay for Testing Virus Stability
The wild type virus AcMNPV E2 was passaged repeatedly at a high or low virus dose in Sf21 cells. The cells were observed at each passage for the appearance of the FP phenotype. For the high dose, 100 µl medium was taken from the culture 5-days post infection, and transferred to a new culture (T-25 flask with 1.5x106 cells). For the low dose, 1 µl of medium was transferred 5-days post infection from one culture to the next (T-25 with 1.5x106 cells). For passaging at the high dose, the FP phenotype was detected at passage 5. By passage 13, no polyhedra were detected at all. For passage at the low dose, the FP phenotype could be seen at passage 11, but was not obvious until passage 17. Passage 17 at the low dose was equivalent to passage 8-13 of the high dose treatment.
We were unable to get good infection of the High Five Tn5B1-4 cell line with AcMNPV E2. The reason for this result is unclear. Our current plan is to use Sf21 cells for testing the recombinant baculovirus for stability on repeated passaging in cell culture.
Sf21 cells infected with wild-type virus (passage 1) or virus with the apparent FP phenotype (high dose treatment, passage 18; low dose treatment, passage 18) have been fixed for examination by transmission electron microscopy. Fewer polyhedra were present in cells of passage 18 of the high virus dose treatment than in the wild-type virus passage 1. Mutation of fp25K results in lack of occlusion-derived virus within the polyhedra that are produced. These methods will be used for examination of the recombinant virus with the stabilized fp25k gene.
The FP mutant phenotype can be detected in Sf21 cells within seven passages when a high dose of virus is used. This information will be used for testing of the stabilized virus for stability on repeated passaging. The wild type virus will be passaged at the same time for comparative purposes. We have constructed a modified fp25k gene with all 13 TTAA transposon target sites removed. This modified gene will be inserted into AcMNPV E2 in place of the native fp25k, for subsequent stability testing. If successful, use of a genetically stabilized virus will allow for cost-effective, in vitro production of baculovirus insecticides. These insecticides will help to mitigate the environmental contamination associated with the use of classical chemical insecticides.
We plan to construct a recombinant baculovirus that expresses the modified fp25k gene in place of the native fp25k, and test the recombinant virus for stability on repeated passaging in cell culture. The wild-type virus will be passaged at the same time for comparative purposes. We expect that the removal of the putative transposon insertion sites from fp25k will result in stabilization of the virus on repeated passaging in cell culture. If successful, use of a genetically stabilized virus will allow for cost-effective, in vitro production of baculovirus insecticides. These insecticides will help to mitigate the environmental contamination associated with the use of classical chemical insecticides. The plans for 2005 and 2006 are presented below.
Plan for 2005:
- April - June: Clone PCR product into baculovirus transfer vector.
- July - October: Construct and amplify recombinant baculoviruses, and check the genomic structure and sequence of introduced fp25k gene.
- November - December: Initiate first replicate of repeated passaging of stabilized virus, alongside wild type AcMNPV E2. Supply modified virus to Dr. David Murhammer for testing in the continuous bioreactor.
Tentative Plan for 2006:
- January - May: Conduct second and third replicates of repeated passaging of stabilized virus.
- January - September: Analyze samples from the three replicate passages will be analyzed by:
- Protein gel to visualize the amount of polyhedrin protein present in cells in alternate passages.
- Western Blot to determine the amount of fp25K protein (fp25K antiserum), and budded virus (GP64 antiserum) present in cells and supernatant, respectively, for alternate passages.
- TEM to examine polyhedra for the presence of occlusion-derived virus.
- In the event that genetic modification of fp25k results in the stabilization of virus in cell culture, we will conduct bioassays to determine whether the infectivity of the virus has been altered by the modifications to this gene.